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Primary Cell Culture - Science method

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Hi Everyone,
I am culturing the monocytes derived from the PBMC of the patients blood samples. After two weeks of the culture growth, some of the cells appeared as shaded (or in background looks like covered by a layer of something else), [pic 1- day 1, pic 2- day 6, pic 3- day 13 and pic 4- day 21] imaging at 0.4x.
Is the cells need passaging? If yes, I am worried weather the these cells will be adhered again or not. But as per the protocol, no need to do the subculture.
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Hi,
Then it is absolutely normal for your cells to adhere to the culture flask. If you're still maintaing them then I would suggest you to split them in 1:3 ratio to be on the safer side.
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Hi Everyone,
I have an issue with change of culture media color. I prepared culture media A and B. After few days, color changed to pink (B). Any possible reason for the same.
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In this case, the color change is due to an increase in pH and not contamination.
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Hi Everyone,
I am culturing the primary cells (Monocytes) from the patient blood samples. During the culture (2-3 weeks), I have seen the long tape shape black color filament (1 or 2 in number). Is it a type of contamination? How to overcome this?
Thank you in advanced.
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Doesn't seem like a contamination, but more of a poly(propylene)fiber filament, a result from plastic injection of pipette tip during production. We usually ignore it, through centrifugation it will disappear, eventually. Else keep an eye on the tip, if one showed with filament seems going to detach, please discard it and choose a new one.
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Hi Everyone,
Should I add L-glutamine during media preparation if the culture media (RPMI 1640 with l-glutamine) already contain the same?
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I aslo think that checking the purchaser information for cells/media would help. And, addition of L-glutamine to medium already containing l-glutamine is mostly not necessary.
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I am wondering if I have primary cell culture (iPSCs/bone marrow-derived macrophages) and immortalised cells (HeLa/U2OS) but only one CO2 incubator in my lab, could I keep them in the same incubator? Also, may I use the same biosafety cabinet for them, with proper ethanol sanitisation and UV? I am worried about the risk that primary culture would be "taken over" by the immortalised cells, such as HeLa.
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Hi Adenine Koo,
You can use the same CO2 incubator and biosafety cabinet for working in both primary and immortalized cell lines. However you should use special precautions while working with both the cells. Do not use both the cells together while working in biosafety cabinet and do not open the caps of the flasks at the same time. Sanitize all the plasticwares packs and surface with ethanol.
Best of luck.....!!
Thanks
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I am isolating and culturing the monocytes derived from patient blood samples. The following step followed-
1. Blood + PBS (1:1) mixed and topped up on the histopaque layer. Centrifuged at 400 g, 30, RT.
2. PBMC layer separated and washed with cold PBS by Centrifugation at 400 g, 10 min, 4C.
3. Cell pellets washed again two times with cold PBS by Centrifugation at 300 g, 10 min, 4C.
4. Cells incubated with culture media for 30 min.
5. After incubation, media changed with fresh media and cultured for further growth. (Image Attached)
6. After 24 hrs, media changed with fresh media as per the protocol.
Image taken which shows many small cells other than round monocytes (Attached). What are these cells and how to remove it?
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There may be some red cells in there. You can add a 30 second water lysis step. Lyse cells in water and then add an equal volume of 2x pbs. There looks to be some dead cells and debris so you may also want to wash the plate with PBS after the plating step. If you are still having problems you may want to look into a monocyte isolation kit like Miltenyi.
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Dear researcher all,
I'm looking for best protocol for primary culture of colonic or cecum intestinal epithelium cells (IEC) from mice. The strain of mice is C57BL/6. Does anyone know how to culture IEC from mice and successful experience?
Best regards,
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Hi,
Let me know if your experiment worked and if possible please send me the detailed protocol. I am trying to set up a primary culture of intestinal epithelial cells from mice.
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Kavitha Jade Brunner it sounds to me like you're doing the right things regarding the water in the water baths. As for cleaning surfaces, using the bleach wouldn't cause contamination, it can just wear down on the surfaces over time.
Disinfecting the scope and surrounding areas as best you can will likely help quite a bit! I'd love to hear if it helps as I am quite curious as to your contamination source as well, now.
I'm not sure why you don't filter media, but perhaps there is something about these cells/this media that I'm unfamiliar with, which is certainly a possibility! Since you're using antibiotics, it can be good to regularly test for mycoplasma as well if you don't already. Though you may not have visible contamination in all your flasks, contamination can sometimes be masked by antibiotics. It seems to be a point of contention among scientists (much like "a-POP-tosis" versus "a-PUH-tosis" haha) but I personally prefer to culture cells without antibiotics. If your aseptic technique is good (sounds like yours is - your source of contamination is likely from the dissection conditions), there's really no need for antibiotics in my opinion! Your PI may vehemently disagree, but that's just my two-cents. Good luck and feel free to keep me updated as to whether the scope disinfection helps!
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On pregnancy day 18 we sacrificed one pregnant rat and we isolated the Hipothalami of the embryos (there were approximately 15 pups). All the Hypothalami were collected in one test tube. They were then further processed.
The isolated primary neurons were then seeded on a 6-well plate (3 Mio/well).
What is the N-number in this case?
Is it 1, since all the embryos derived from the same mother?
Or is it 6, since there are 6 wells?
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The n = 1 with 6 technical replicates because you processed all of the embryos into a single tube before plating. If you had processed each embryo and plated the resulting cells separately your 'n' would be 15.
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Hello everyone,
we have transformed a PDAC 3D organoid into a 2D culture. After a few passages, we saw those vacuole/lumen-like structures occurring (seen in the picture). At the same time, the proliferation rate (measured via confluency by phase-contrast imaging) slowed down.
Has anyone seen this before or has an idea what this might be? What could be the reason for it?
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Hi Vincent,
You can test the cells for senescence markers using a number of kits.
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I have been trying to isolate and culture mouse bone marrow-derived macrophages but have been getting a much lower yield than I expected. After isolating cells from mouse femurs and treating with RBC lysis buffer, I plate about 12 million at 3e5 cells/cm2 in DMEM with 10% FBS, 1% penstrep, and 25ng/mL human recombinant M-CSF. I use non-tissue-culture treated 6-well plates. I replace the media twice over the course of a week. After a week, I wash twice with PBS and then detach the cells with Accutase. My yield last time was about 80,000 cells after culturing.
I think part of the problem may be that the cells aren't attaching to the plates and so they're being washed away before I can collect them. I've never dealt with primary cell culture before but I was expecting that if the myeloid cells differentiated into macrophages, they would adhere more firmly to the plate.
I would greatly appreciate any advice anyone might have about how to better culture these cells.
Thanks!
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To easily and rapidly lyse red blood cells adhered to macrophages,
I use an ACK solution (made of ammonium chloride is a buffered solution, prapared in house or commercially available, too), for 5 min. at room temperature.
Macrophages can resist such treatment with ACK but not the red blood cells.
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Hello!
I isolated some T-cells with some very interesting TCRs from primary cultures. I sent them to Genewiz to get chromium single cell TCR sequencing done, however the sample viability was super low. I sent them 8 more vials so that they could do a dead cell removal and then isolate the live population and perform single cell sequencing on the remaining cells. The sequencing results show that whatever is left over after DCR is most likely another contaminant cell type, not a TCR. I now only have one vial left, so whatever I choose to do next is very critical and essentially has to work the first try.
At this point I dont care about chain pairing, I can piece that back together afterwards by trial and error, I just want to get some data from these cells. I was wondering if anyone knows if I can thaw my cells directly into RNA later and then do either normal NGS or another single cell sequencing method to get any info on the TCR sequences? Should I just amplify the TCR regions on thawing with some kind of primer pool and then send that for NGS? In general, what's the most robust process for getting out TCR information from low viability samples?
Some other notes:
1. I didnt personally do the T cell isolation but my thinking is they were pretty much exhausted at the time of freezing which is why we have viability issues on thawing
2. They were frozen in 10% glycerol + 10% FBS in a Mr Frosty at -80C and then maintained in LN2 and shipped on dry ice.
3. Observed viability is ~30% on thawing however this could just be the contaminant cell population....
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Definitely a fair assessment, Ive made other samples that have similar properties as this, but none of the other samples Ive generated have responded with as much vigor as this sample.
Definitely will invest in generating another panel of T-cells but from what I can tell so far, this sample had a particularly rare phenotype that i may or may not see again in a relatively limited panel size. every once in a while I remember I have this last vial and wonder if I can do anything with it, in the end its always for the birds...
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Hi All,
I am working with digested fresh tissue (Pancreatic cells) and I am literally having a contamination which is seen as a black swimming dots all the time.
I digest my specimens and I culture them into 12 well plates. After checking them under microscopy I can see these black dots that are kind of stable and not moving, which make me think that they are only debris (some cells are dead and some are alive).
After a week or even more, when the cells are bit confluent, these black dots appear clearly moving. Despite that, the cells look healthy and happy.
I am always very cautious during cell culture experiments. I always check my medium before using it, adding antibiotics, sterilizing the hood...
I would appreciate if anyone could help with this matter.
Thanks in advance
Fouzia
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For primary cells isolated directly from tissues, or their early passages are at a particularly high risk of contamination as the dissection process may promote contamination from the natural commensal flora in certain tissues and any subclinical infection. For cells isolated from primary tissue, it is necessary to use antibiotics in the primary culture. Antibiotics should be removed as soon as possible, and the culture should be tested for mycoplasma after at least two passages in the absence of antibiotics.
Please refer to the link below for preventing contamination of primary cell cultures.
Hope this helps.
Best.
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hi! I'm working with isolation of primary cancer cells using explant culture technique. Usually the cell type I'm dealing with cells which adheres within 7-10 days. But now the cells are not adhering and are still suspended in media. I've tried to reduce the surface area, checked carbon dioxide levels and my control cells are growing just fine (means my media reagent, FBS and supplementation are correct). Also, want to add my protocols are optimized and have been using it for over a year now. I have no other cell types in my incubator.
I wonder if anyone can help me to how to make sure the cells adhere. Any suggestion will be highly appreciated.
Thanks in advance!
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Dealing with primary cell culture can be tedious sometimes and of course, they are not easy to deal with.
We specialize in producing high-quality primary cells. You can connect with us at info@kosheeka.com to know about primary cell culture or you can visit our website https://kosheeka.com/ for further details
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My lab is working with primary cells from the patient and we would like to check if our cells are infected with HIV, HBV, HCV, etc by PCR. We would like to know how many cells do we need for this. The thing is that we don't have a lot of cells so we would like to use as little as possible.
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depend on the lower limit of detection (LOD) of the PCR, the viral load, and the stage ( or clinical feature) of the patient
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I want to isolate pancreatic islets from hfd treated mice to study the expression of few proteins in western blot. will not do primary culture from the isolation. Please mention the protocol and should I need to isolate the islets immediately after animal sacrifice or can be done after few days or months after storing the pancreatic tissue in freezer?
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Diana is right, you have to isolate islets immediately. In our lab we use the protocol that we just updated in this publication to isolate islets.
For the WB: I collect about 100 islets that are similar in size and wash with PBS, spin and collect PBS. Add 30uL of SDS sample buffer and boil for 5min (basically you are lysing your islets in the running buffer to avoid dilution). Load 12-15uL on a western blot lane (I use Bio_Rad mini gels.) If you use islets of similar size, I don't have problems with unequal loading.
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I would like to obtain primary culture from mesothelioma and ovarian cancer samples. Which concentration of collagenase (unit/ml) can I use for the dissociation of these tissues? And incubation time ?
Does anyone have an efficient protocol?
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@benedetta colmegna
Hi, I have the same question. Could you let me know the protocol you finally ended up using?
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A few months ago we carried out a primary culture from murine adipose tissue aiming to isolate mesenchymal cells. Two days after the primary culture was stored in DMEM + HAM F12 the culture medium turned yellow. The protocol had been used in the past, but this was the first time turning yellow and in such a small amount of time. What could have been the reason for this phenomenon? It is worth mentioning that along with mesenchymal stem cells, muscle cells, and cells of the immune system were also observed. Also, no microbial contamination was found.
Thanks in advance.
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if your media has changed color but not turbid (indication of bacterial growth), your cells have exhausted the media. Phenol ref in the medium is added to see the physical appearance of culture so as to change/replenish the medium.
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We are growing amniotic mesenchymal stromal cells in culture. The cells are isolated from a term placenta, delivered via C-section, using collagenase. We culture the cells in 10 % FBS DMEM (0,1 % Gentamicin). The serum we use is not filtered. The isolation is carried out under strict aseptic technique.
I have attached a photo of the culture 16 days after isolation (passage 2). Numerous black dots can be seen (some I marked with red arrows).
We also carried out a DAPI stain (see attached), but it was negative for Mycoplasma contamination.
Are the black dots from contamination with bacteria or fungi? Or are they simply cell debri? The cells do not grow very well, we are worried the black dots could be connected to the stunted cell growth. Also, the cells adapt a wide shape, instead of the slim fibroblastic morphology they should normally maintain.
Today we also placed samples of the serum and the medium we are using into an incubator (aerobic and anaerobic conditions), to see if the black dots will appear or not. I will post updates 5-7 days later.
Any ideas will be appreciated. Thank you!
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You may also refer to the article below.
Best,
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Dear all,
I need to culture whole blood in a 37°C incubator for 24h prior to RBC lysis and analysis.
If blood is sampled in Na-Heparin tubes, i think that it is required to add additional Na Heparin to the cell culture vessel to prevent clotting during the 24h culture.
Have someone already done that ? I read somewhere that 3 IU/mL Na Heparin is enough. Do you agree ?
Thank you.
Best regards.
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If you have drawn the sample in the sodium-heparin tube then you need not to add further Na. Sodium in the tube is enough to stop coagulation.
Even if the coagulation will occur it will occur in 30-40 minutes. Check with control sample and then think of adding more Na to the tube.
Hope this is helpful.
Good luck.
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We have used the same isolation and expansion protocols for the generation of PMEFs for many years and never had any major issues. We recently changed our batch of FBS and now we are seeing massive cell death following the first passage. Most of the time its apparent the next day, but we have had two batches where they have looked fine the day after passaging only to have the majority detach the following day. If the surviving PMEFs are left to grow we don't see massive cell death with subsequent passages, so i'm thinking the survivors have adapted to the media (specifically the FBS).
The isolation media is pretty basic; high Glucose DMEM, 15% HI FBS, 0.1mM 2ME, 50U/mL Pen/Strep and 2mM Glutamine. We culture in this media for two days then passage into 10% HI FBS, all other components stay the same.
Historically our FBS testing has been focused on how our mES cells perform and they grow fine in this batch of FBS.
We would prefer not to have to use two different batches of FBS for culturing our cells to avoid mixups and additional expense, so before we start looking into new FBS does anyone have any suggestions/advice.
Thanks
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FBS may vary in quality in terms of growth factors, amino acids and other components from batch to batch. Initially the cells may not show the effect of the new batch of FBS because they already have stored the necessary growth requirements for their growth from the previous batch of FBS. Once these stores are depleted the detrimental effect of the new batch of FBS if any becomes visible because the cells will be utilizing the new batch of FBS.
So I suggest you culture your cells in the new FBS batch alongside the present usual batch of FBS in which the cells are growing fine. Compare various parameters such as cell viability, morphology, etc. just to see that the new batch of FBS does not have any detrimental effect on the cells. If everything works fine you can grow your cells in the new batch of FBS.
I hope this helps.
Best.
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Hello,
Recently I thawed couple of cell lines, RPMI8226 and JJN3 and they both didn't do well during my first passage.
I thawed the vials in 37 deg water bath for couple of minutes. Transferred the cells with media into a conical tube. Centrifuged the tube at the lowest speed for 5mins. Aspyrated the supernatant and resuspended the cells in 10mL media in T-75 flask for 3 days.
I use the following media:
Gibco RPMI 1640 with L-glutamine + Penstrep + Glutamine + 10% FBS
My percent live cells was very low when I checked them 3 days later. I am thinking of using 20% FBS next time I thaw cells. Is there anything else I can do differently to keep the cells alive?
Thank you!
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Hello,
As Joseph mentioned, you have to be careful for how long you´re warming the cells, thawing freezed cells has to be a fast process, since DMSO is toxic for cells and can be the reason why you have low percent of live cells. Remember to check for any thawing specifications regarding your cell lines with your supplier.
Basically, both Freshney´s Culture of Animal Cells textbook and Thermo Fisher Scientific´s webpage recommend the following general thawing protocol:
1) Remove the cryovial from liquid nitrogen or -80°C freezer and immediately place it in a 37°C water bath. 2) Swirl the cells until you see a small bit of ice in the cryovial (this shall take about one minute). 3) Transfer the cryovial to a laminar flow-hood, open it, and transfer the thawed cells to a centrifuge tube. 4) Add pre-warmed medium appropriate for your cell line in a dropwise matter. 5) Centrifuge the cell suspension at approximately 200 g for 5-10 minutes (this vary with your cell line, I recommend you to check). 6) Decant the supernatant, resuspend the cells with medium, and transfer to a culture flask.
Note: some cells can be thawed without the centrifugation step, you can dilute the cryoprotectant with fresh pre-warmed medium, but you must change the medium the day after thawing.
Suggestions: make sure to work fast but aware of the steps of the process; while thawing the cells in the water bath make sure not to submerge the cryovial since this can increase the probability of contamination; try not to thaw cells that were freezed a long time ago, since viability can decrease; remember to always work aseptically; double check the label of the cryovial to make sure of the identity of the cells; plate thawed cells at high density to increase recovery.
In case you want to read more about thawing, Freshney´s textobook is very useful: Freshney, R. I. (2006). Basic principles of cell culture. Culture of cells for tissue engineering, 3-22.; you can also visit Thermo Fisher Scientific website, which has a video and a guideline regarding the thawing process: https://www.thermofisher.com/cr/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/thawing-cells.html
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This is a long-shot question. I have some methanol-fixed tissue neural tissue. I'm wondering if it would be possible to get intact single cells from the tissue once rehydrated. I was thinking mechanical dissociation with trituration. If this were fresh tissue, I would use a protease like papain first to help the dissociation, but since this tissue is methanol fixed, I'm concerned that (1) the protease won't work (2) it may work but it will go intercellularly and mess up the internal proteins.
Is there any precedent for dissociation of fixed tissue?
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Sarah Hélène Merkling - vivoPHIX sounds really interesting, have you used it yourself to process tissues + dissociate for scRNAseq? Would you mind sharing your experience with it? Thank you!
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what kind of media and how to culture it?
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A gel substance like substance as media. It must contain nutrients for the B cells. Should you not stimulate the cells from bone marrow? Maybe from small age. The stimulation could be more of human.
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I have been trying to culture primary fibroblast cells from mouse skin for awhile now according to the following protocol:. I want to use these cells for co-culture and measure their activation to myofibroblast via a-SMA and Collagen Type I immunostaining. However, I consistently encounter the issue in which my fibroblast cells express a-SMA at steady state even before the addition of positive inducers like TGFb. I know fibroblasts are sensitive to mechanical tensions and hence tried growing the cells on:
  • glass chambers
  • glass chambers coated with Collagen Type I
  • glass chambers coated with 1% gelatin
  • ibidi polymer chambers with ibiTreat
All these conditions results in a-SMA+ fibroblasts. I am not sure what is the issue here. Does anyone know why?
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Its really a very simple answer!
1. Serum which you use as a survival supplement is an alarm signal; it indicates tissue injury (serum is the liquid component of clotted blood).
2. Serum proteins adhere to the culture vessel surface, providing cell attachment ligands (recognised by cell surface integrins). Integrin engagement, in turn, facilitates cytoskeletal organisation (aka stress fibres), of which SMA is just one element. Given TC plastic is rigid, this provides mechanical resistance, that stabilises the actin cytoskeleton.
You see, its really simple, once you look carefully at what is in your system!
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Currently, I am looking for a solution to develop chemotherapeutic drug resistance in a primary cancer cell line. But after a quick look at the literature, it is indicated that the management of acquirement of drug resistance takes plenty of time, more than eight months! Is there any convenient method to subculture chemoresistant cell lines in a short time? Or any other suggestions rather than eight months interval with an increasing dose of chemotherapeutic agent?
Best regards.
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Dear researchers, thank you all for your contributions. Dear Mohana Krishna Gopisetty, the method you have suggested is time-efficient and problem-solving, I appreciated that. However, the cancer type we are working with is a little bit struggling. Our drug of interest is one of the members of the chemotherapy regimen which consists of other different drugs (we can say that it is given as a cocktail). Also, the relative survival rate of patients is low, diagnosis with specific drug chemoresistance takes time. (but we know that chemoresistance happens - under investigation)
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Hi all, I'm currently trying to isolate oligodendrocytes from GA62 (term is GA70) guinea pigs and am having a bit of trouble getting them to survive. I take the tissue and digest with papain and then plate at 10million/T75 flask in DMEM with anti-anti and 10% FCS. This step is fine and they reach confluency around DIV 10-12. I preshake them for an hour to remove microglia and then shake overnight, and then plate in a petri dish for an hour in the incubator to remove any residual glia. I plate them at 20,000 cells/well in a ornithine coated 24 well plate in a mixture of DMEM/apo transferrin/insulin/sodium selenite/D-Biotin/hydrocortisone and 20ng/mL of PDGF-AA and bFGF. I've added 10uM/well Ara-C on day 2 after plating and did a complete change to remove it 3 days later.
I then remove the growth factors and replace with T3.
I've attached some photos from DIV 6 after plating and before growth factor removal but they don't look great. Can anyone offer any suggestions? I was wondering about adding NT-3 into the mix, or maybe not leave the Ara-C in for so long? Any help would be appreciated :)
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One very basic tip depends on the % CO2 you are using. Without serum, DMEM has a bicarbonate concentration that relies on 10% CO2. If you want to remain at 5% CO2 go to a DMEM/F12, 50/50 medium, which has the appropriate bicarbonate for 5% CO2. Past that, I have not worked with microglia, but trying less time in Ara-C is something to try. Good luck.
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Dear All,
I work with human MSC primary culture. I and my colleagues have observed suspicious contamination in our culture (different MSC sources, two different incubators). Can you help to identify the kind of contamination? What should we do?
That concerns us a lot - we observed that cells did not want to attach to surface after passaging. we noticed worsen morphology of our cells, changes in housekeeping genes expression in PCR.
What have we done so far?
- colometry assay for Mycoplasma - no detection
- PCR - no detection,
- microbiologic tests for molds and yeast - no detection,
- always filter 0,2 um medium with plate lysate before use,
- used antibiotics: firstly mix - penicillin-streptomycin-amphotericin, secondly - double the concentration of that mix, then - pure amphotericin; no results,
- before work, sterilize hood with UV; every day at the end sterilize room with UV; using alcohol and anti-mycoplasma cleaning tissues and liquid detergent,
Can someone help us?
Photos: primary culture of WJ-MSC in magnification x10 and x20. Black dots may be bacteria (they were slowly moving).
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Dear Agnieszka!
If you have done all these procedures and did not find contamination, according to the photographs, these may still be dead cells due to the type of apoptosis, when small apoptotic talci are formed. Based on your description, cells do not grow, do not attach, but die. Apotosis is a physiological, programmed type of death, and therefore your cells do not like any factors. Check media, serum, supplements to ensure all reagents are fresh. check the culture dishes (dishes, plates, vials), you may have used other manufacturer's dishes with different adhesive properties. Primary cell cultures like to grow on highly adhesive plastic.
One more thing: Your cells can just get senescence. In this case, their adhesion also decreases, the expression of surface markers changes, and increased death is observed. In this case, it is better to replace the culture, to defrost new cells.
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We're just starting to isolate pancreatic fibroblasts from healthy and a tumorigenic pancreas but it does not work very well. We tried freshly isolated and also frozen tissue and use a collagenase IV digestion. Additionally we add FGF to the media.
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Miriam Stölting Hello, we have been trying to isolate fibroblasts from the fresh human pancreas but haven't had a bit of luck. May I know the process and the medium you use in the process? Thanks.
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I would like to examine effects of particular drugs on D1 and D2 receptors using culture, but wondered if this is possible as people tend to look more at TH-positive cells. Thanks!
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Hello
I not exactly sure what aspect of D1 and D2 receptors you are interested in. For example, protein expression levels, mRNA expression, cellular location etc, or simply to mark particular types of cell. It sounds like you are interested in labelling D1 and/or D2 expressing cells, perhaps with a goal of quantifying how many there are, or some change in distribution or cell morphology.
There are plenty of companies selling anti D1 or D2 antibodies that may work for immunohistochemistry and I have seen publications looking at least D1 expression in rat cultures from the striatum (PMID: 11320256). My experience of performing this type of staining is to just try it as antibodies can be hit or miss. Unfortunately, they are expensive for just a pilot and you may try asking the company for a sample. Also, on this issue I find that staining in cultures can be particularly error-prone with regard to achieving a genuine signal as there is not as much background material to compare to. With this in mind, some kind of control is a very good idea, such as antibody blocking, or even better, testing the antibody in tissue from D1/D2 knockout mice.
Aside from immunohistochemistry, there are many other ways you can look in on D1 and D2 depending on your question, e.g. Western blot, RT-PCR, receptor binding etc. There are also mice with fluorescence-tagged D1 and D2 receptors (e.g. PMID: 28436559), though clearly introducing them into your study may be a much more involved and costly affair. That said they deliver an excellent degree of confidence in the legitimacy of the expression.
Measuring expression of TH is as you say common. I would expect it to be far easier than D1 and D2 by immunohistochemistry, but it is quite a different but related question. Again, I am not sure what your goal is, but measuring TH is commonly used for identifying dopamine neurones, but do remember that TH is also involved in the production of norepinephrine and epinephrine so depending on from where you are taking the cultures, you may need to consider this.
Good luck!
Niall
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I'm culturing primary cells from equine bone marrow. The possible contaminant is in the upper side of the flask, not in the bottom where are the cells and the media. What it can be and how can I get rid of it without having to discard my flasks? Thanks!
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Have you sold this problem and what is the source of contamination? We have the same problem in our lab..
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Hi there,
I’m currently growing primary neurons and they're confluent and confirmed with ICC staining. I’m using 24 well plates with coverslips coated with PLL, and have tried both the RNeasy mini and micro kits and have managed to get a total of 7ng/ul RNA from 24 wells. I’ve been lysing the cells in the wells with RLT + B-mercap. Any advice is very appreciated!
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Ok you should probably have more than 84ng of RNA in total. I'm a little confused - you lysed the cells and then spun down the media? The media should be removed before addition of the lysis reagent. My protocol for RNA extraction from cultured neurons is roughly:
1) Remove and discard all media
2) Pipette required volume of lysis reagent into each well
3) Gently rock/tilt plate to ensure lysis reagent is in contact with all cells
4) Insert pipette tip into first well, vigorously stir the lysis reagent in the well and pipette up and down to make sure all cells are lysed and contents suspended
5) Transfer all liquid from well into a microcentrifuge tube
6) Repeat 4 and 5 for every well
7) Vortex all tubes thoroughly
8) Proceed with RNA extraction according to kit instructions.
I would not spin down the samples between collection and loading them onto the columns unless the kit instructions say to do that.
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I have been working on astrocyte from primary glial cell culture and I want to observe calcium influx after several treatment. I know once Fura-2 AM in the cell, intrinsic esterases hydrolyze the ester bounds and trap the dye into the cell. Therefore loading astrocytes with Fura-2 AM and fixing it by 4% PFA on the coverglass should have work to see the calcium influx happened just right before the fixation, isn't it? Or is the fixation processes by 4% PFA will destroy the membrane and cause Ca2+ fluorescence to be lost?
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no I never worked with fixated tissue extrinsic fluorescence. sorry, i misanterstood you problem, cannot help further, thanks for your answer to my coment, Vera Lima
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Hi everyone,
I have been trying to get a gigaseal from primary cell-culture cells from rat’s DRGs, but without success. I don’t know exactly what the problem is.. I have tried after 2 hours form getting the cells, after 24 hours, and 48 hours, I have changed the glass used to fabricate the pipettes, (I tried both thin walled glass and thick walled glass), I have tried with different pipettes resistances (from 2MΩ to 10 MΩ) but without success.
A PDF that I prepared is associated to explain what I have done, containing screenshots of what I get in the “SutterPatch” Program, and picture of the pipettes I used.
Do you have any recommendations or a solution to help forming gigaseal ?
Thank you in advance!
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Thanks very much, we will try this and see what we will get
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Hi everybody!
Last year we started the attempt to immortalise cells derived from human prostate cancer tissue (RPE material). One of the derived cell lines is very different from the others. On a morphologic level they look a bit like fibroblasts but lack the characteristic pattern in higher confluency and all tested fibroblast marker, too. They lack also protein expression of AR and PSA but show expression of AR-RNA (ca 1/10 of the 22Rv1 level) and are hormone-insensitive. We tested them for stem cell and neuroendocrine marker plus EpCAM and KRT 5, 8, 14, 18 and 19. They are negativ for all of them. We also performed an antibody staining for smooth muscle actin which was negativ as well. By in vivo and in vitro experiments we found no tumorigenic potential of the cell line.
Is anybody able to make a suggestion for this cell type or further marker?
Thank you in advance.
Simon
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I seems like it's PC-3 !
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Hi,
I am studying purified mice CD4 T cells, CD8 T cells and DCs etc proliferations in vitro labelled with CFSE. But total splenocyes are NOT showing proliferation. How can I observe splenocytes proliferation in culture. I am using both the ways for stimulating splenocytes either by specific antigen or anti-CD3 & anti-CD28 or ConA stimaution.
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Stimulation with CSA/ PhA and kept in CO2 for 3 -4 day with RPMI+15% FCS. after that at least 50-65% proliferation will found.
It's very difficult to handing also during this period contamination of culture is very high changes because of primary culture.
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Hello fellow researchers,
In one of my experiments, I need to identify cells in G0 phase in my primary cell culture. It will be very helpful if anyone have any experience with such experiment and can suggest any marker based or flow based assay.
Thanks in advance
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Dear Rahul Kumar by using DNA dye you will be able to separate G0/G1, S and M phase but if you add RNA dye as well, you will be able to separate G0 and G1. Attaching you an article showing protocol and gating for the same
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Hello everyone,
I have been trying to establish primary rat DRG neuron culture for a couple of months already. I am using the protocol I found somewhere during the Google search, apparently coming from materials for Biology classes ( https://nanopdf.com/download/preparation-of-rat-dorsal-root-ganglion-neurons_pdf# ) with some modifications:
  1. I use P1-P2 rats instead of embryos
  2. I coat coverslips with Matrigel and culture the cells in 6-well plates
  3. I added a collagenase IA digestion step (60 minutes) to get rid of the capsule and I digest with trypsin only for 15 minutes.
  4. For mitosis inhibition I use a mix of 5-fluoro-2'-deoxyuridine (10µM), uridine (10µM) and cytosine-ß-D-arabinoside (1µM). It is added at DIV2, DIV4 and DIV6.
The rest is following the above-mentioned protocol.
My problem is, even though I managed to get quite a number of cells from the preparation, they start to die off very quickly (around DIV3-4 - pics attached). I noticed, that even if I restrict the initial seeding surface to around 1 cm^2, when the restricting ring is removed from the culture, the cells disperse on the whole glass. Do you think restriction of the surface for a longer time would be helpful? Or could the mitosis inhibition be an issue? Or do you see anything else that could hamper my trials?
Thank you very much for all your answers - I am a total newbie to cell culture and in my lab not many people deal with neurons in general.
Best regards,
Marta
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Hello Marta,
The survival of P1-P2 DRG neurons will depend on the cell culture media you use and the presence of trophic support. Adding NGF to this cultures will help you to keep these neurons alive, but may not really fit your experimental protocol. More mature DRGs (e.g. P30) are less dependent on trophic factors for the survival.
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Hi, everyone i am going to use FCS- depleted exosomes From Thermofisher, for primary cell culture of bovine oviduct epithelial cells , and i am wondering if i need to inactivate it befoe its use
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Judging from the product description the exosomes are removed by ultracentrifugation of the serum without heat application. In this case, natural IGG's are still present in the serum and its heat inactivation may improve your culture growth. Even if you perform double heat inactivation of the serum it will not render it's useless slightly degrade it's quality at worst. You can also prepare a small amount of the media with heat-inactivated serum and without and see which is best for your cell's growth.
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We have been experiencing contamination in our macrophage primary cell culture for about a month now. We have tested all media and reagents used in the isolation process, used all new reagents, cleaned the incubators, hood, and any other equipment used. It appears to be resistant to penicillin/streptomycin and gentamicin. The media doesn't turn yellow, but there appears to be what looks like yeast floating on top the solution. They are the small, white squirming cells. Any feedback is greatly appreciated!
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Hi, I'm also encountering such problem in my cell culture. So the whitish cells are bacteria or just debris? Thanks.
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I am planning for anterior pituitary cell culture for 4 days in medium containing estradiol. I do not know how long can estradiol have its effect in culture cells. Can you help me? Thank you.
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Hi all. I am planning you using beta-estradiol on agar plates for yeast. Any idea how stable will it be there? Thanks
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I am trying to separate mouse/human gastric/intestinal mucosa, submucosa, muscle, serosa and want to do the primary culture for each tissue.
Since the layers are thin, are there any ways to separate them layer by layer?
Or are there some protocol videos?
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Plesae type your question in google. You could find a lot ad answers then, you have to select easy one.
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Hive mind:
I am trying to grow cells from bursa taken from chicken embryos. I am using a protocol very similar to the one described here: 
I have two main issues, which are possibly caused by the same problem. 
1. I am getting an ABSURD amount of red blood cells. I want to try a lysis buffer on my sample for my next try.
2. The viability of the cells is very low (around the 8%).
Does anyone have any experience with this kind of primary culture and can offer tips or protocols that might help? 
Thank you
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Dear Ori
I am trying to isolate B lymphocytes from bursa of fabricius for studying lymphocytes proliferation assay. After seeing your answers and tips that mention cells with low viability, I wonder whether it is feasible for lymphocyte proliferation assay because usually this experiment takes 2 or 3 days. I am afraid all B lymphocytes would be dead, limit references I found that involving this...
Wish for your reply, gratefully!
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I am doing a primary culture, and I have some problems while culturing the cells.
When I culture the cells from primary to passage 1 and 2...
lots of cells differentiate and stop dividing before I get a sufficient amount of cells.
How can I prevent primary cells from differentiating and divide well?
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I use primary keratinocytes (KC) from the oral cavity, and experience that several factors can start early differentiation. Saurabh Mandal and Yakun Wu mentioned several factors:
- area (ours KC do well in T25 but not bigger flasks)
- number of cells per area (to few KC will start early differentiation)
- medium (to much calcium starts differentiation)
- splitting (never grow to confluence; check media used to split, return quick back to right medium; our KCs do well for 6-7 passages but start to differentiate after)
- age of the donor (we have bad experience with elderly donors)
- site of biopsy (KCs from some sites grow better then other sites)
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I'm trying to grow PDX-derived cells from a SCCOHT model and I need them to survive short-term (7-14days) ex vivo
After tumor digestion I platted 7,000,000 cells in a 10cm ULA dish
In the following day they were forming spheroids but the spheroids were aggregating/clumping (First picture)
I filtered through a 40uM strainer and collect the portion that remained in the filter, washed with PBS, added 0.5mL of trypsin for 40s, neutralized with 1mL of 10% FBS media and them diluted in the serum reduced media (Counted with trypan blue and had >90% viability - Second picture)
I repeated this one more time after 3 days but they keep forming these giant aggregates every 2-3 days
I'm unsure if it is worse to separate them into single cells and lose the cell-cell contact or to let them grow in aggregates of spheroids
Does anyone know how to procced in this situation?
I digested the PDX tissue in Dispase/DNAse for 30min, filtered through a 100uM strainer, lysed the RBCs, minimized the debris with Ficoll and them platted in Advanced DMEM + 5% FBS
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Do you want to generate independent spheroids .
Varying concentration of serum with methyl cellulose
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Hello,
I am currently working on a project that requires me to transfect a DNA plasmid (5,600bp) into HVMECs. I want to inquire about available transfection protocols that would yield in high efficiency of transfected cells. I previously tried using chemical transfection reagent, JetPEI but results were very variable. My DNA plasmid has no reporter gene to measure transfection efficiency, and encodes a lncRNA so western blot analysis is not feasible. I am attempting to measure efficiency by looking assessing downstream genes via qRT-PCR. Any suggestions on better transfection protocols would be highly appreciated. Thanks in advance!
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Thank you all for your suggestions. I've considered them all and I will most likely go with AAV transfection. The reason I want to stay away from electroporation is due to the low cell viability and my samples tend to be small.
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Hi,
I am looking for a software that would allow me to record real-time videos of my ciliated epithelia primary cell cultures using an inverted microsope and Canon camera.
Currently, I am using QuickPHOTO Camera 3.0 which does not allow me to record videos (only time-lapse with intervals 13s between images).
Thank you.
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Thank you very much Charles D Anderson
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hello every one, can some one tell me the appropriate and easy to follow protocol for keratinocyte isolation from adult mice epidermis? as I have to do it, but I am  not getting well established protocol?
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For passage experiment make sure the the media is in low calcium by using Chelated FBS. Hope this helps.
Regards
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Can anybody refer me where to obtain immortalized human Schwann cells? I am planning on working with them but I am having a hard time obtaining some. Thanks!
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Have you gotten the cell line?
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I am searching for the protocol of Chromatophores isolation and culture from fish skin. Can anyone kindly guide me regarding the experiment?
Thanks in advance.
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Timir Tripathi , certainly you are aware of the following article
  • A Method for Extracting Pigments from Squid Doryteuthis pealeii by Christopher W. DiBona1, Thomas L. Williams1, Sean R. Dinneen1, Stephanie F. Jones Labadie1, Leila F. Deravi1,2 ,
1Department of Chemistry, University of New Hampshire,
2Materials Science Program, University of New Hampshire
Original 'web location': =
for the following 2 URL's: after pasting the link-address into your browser, delete first the underline character " _ " in between "http _ and s" before you click on "RETURN":
[ http_s://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226083/ , for the original PDF:
http_s://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226083/pdf/jove-117-54803.pdf] , in the pdf:
cf.: 2. Isolating Chromatophore Pigment Granules and
3. Pigment Extraction
and BTW, there exists also a Video of the technique(s):
(NB: quote: "This is a sample clip. If you're new to JoVE sign up and start your free trial today to watch the full video! If your institution has an existing subscription, log in or sign up to access this video." End of quote.
There is an "old" source available (unfortunately only PPV, no warranty for "isolation techniques" contained) ):
Fish Physiology
Volume 3, 1969, Pages 307-353
(NB: same as above = delete first the underline character " _ " in between "http _ and s" before you click on "RETURN": applies to:
http_s://www.sciencedirect.com/science/article/pii/S1546509808601168)
In Vitro Cellular & Developmental Biology
  • Purification of Black Moor Goldfish melanophores and responses to epinephrine, by Carl R. Clark, John D. Taylor, T. T. Tchen, June 1987, Volume 23, Issue 6, pp 417–421 (NB: same as above applies to: http_s://link.springer.com/article/10.1007/BF02623857)
  • Cytophysiology of Fish Chromatophores by Ryozo Fujii
International Review of Cytology
Volume 143, 1993, Pages 191-216, 216a, 216b, 217-255
(NB: same as above applies to:
http_s://www.sciencedirect.com/science/article/abs/pii/S0074769608618768 )
  • Chaplen FW, Upson RH, Mcfadden PN, Kolodziej W. in: Pigment Cell Res. 2002 Feb;15(1):19-26.
Fish chromatophores as cytosensors in a microscale device: detection of environmental toxins and bacterial pathogens.
(NB: same as above applies to:
http_s://www.ncbi.nlm.nih.gov/pubmed/11841070 )
  • Skin Biopsies as Tools to Measure Fish Coloration and Colour Change
P. Andreas Svensson and Helen Nilsson Sköld 2011,
Correct Citation: Andreas Svensson and Helen Nilsson Sköld (2011).
Skin Biopsies as Tools to Measure Fish Coloration and Colour Change, Skin Biopsy - Perspectives, Dr. Uday Khopkar (Ed.), ISBN: 978-953-307-290-6, InTech, Available from: http://www.intechopen.com/books/skin-biopsy-perspectives/skin-biopsies-as-tools-to-measure-fish-coloration-and-colour-chang
  • From: Eberle A.N.: The Melanotropins. Chemistry, Physiology and Mechanisms of Action. Basel, Karger, 1988, pp 210-252 (DOI:10.1159/000415282)
11. Effects of MSH on Pigment Cells https://www.karger.com/Article/Pdf/415282
...confessing / believing, it might be personal 'fortune' to get presented a "comprehensive / universal' protocol for isolation of chromophores ....
Best of luck, WHM
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I am studying neurodevelopment using primary cerebellar granule neurons (CGNs) with a focus on studying morphology. CGNs undergo dynamic morphological changes in vivo and in organotypic slice cultures in vitro but I have not found papers studying morphology in primary cell culture.
What cell coating would you recommend for studying morphology in neurons in general? I've read PEI is good for having neurons remain spread out and unbunched, and I've also seen some protocols using fibronectin. My protocol recommends coating with poly-D-lysine and laminin for promoting neurite outgrowth but if there are other coatings that people recommend I would appreciate having some alternatives as I'm fine tuning my cultures.
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I am culturing primary cells and use DMEM F12 (1:1) cell culture medium supplemented with human insulin and EGF. How long are these components stable for in the medium?
Thank you for your gentle attention.
Regards,
Celina
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1) A study from Sigma on stability of insulin and IGF-1 in cell culture:
" Insulin is degraded in cell culture by enzymes (insulinases) secreted by cells into culture media. In addition, insulin is more rapidly internalized and degraded compared with IGF-I and LONG®R3IGF-I. The extended cell culture stability of LONG®R3IGF-I results in prolonged activity and associated benefits to cell culture. (Graph 5)."
In their conditions, 50% degradation of insulin during cell culture occured after 1.5 days.
2) I would supplement it further with trace elements like selenium as it was done in further versions of F12 media (this way the media will provide all the necessary nutrients/elements - though, the concentrations won't be necessarily optimum):
3) If you don't add any protein to the media (e.g. 0.1 mg/ml BSA or some more indifferent protein), there may be a problem with adsorption and inactivation of the tiny amounts of growth factors on plate walls. That's a real problem when you dilute enzymes - e.g. luciferases - to very low concentrations:
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Is it a problem, if I keep them overnight and replace with growth media next day?
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Cos7 and SH-SY5Y cells were used in my experiments. I found Lipo3000 is toxic when incubation overnight. So I change medium 4 hours after transfection, the protein expression is good. Hope it is helpful!
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Hello,
I have problems with RNA extraction from cells. I work with primary neurons and astrocytes cultures, with 1*10^6 cells for each well (more or less, because some cells die).
Actually, I use the Trizol extraction method. With tissue I don't have problems but with cells the 260/280 ratio is horrible, maybe because of Trizol. I don't know. I use the Trizol protocol and I add DNAse in the last step because my probe can react with gDNA.
I use these samples for quantitative PCR, for this reason I need a pure RNA. How can I solve this problem? How can I improve the ratio?
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First I would recommend to keep right ratio of cells to Trizol reagent. For I mln of problematic cells such as neurons I would use minimum 1ml of Trizol. You can use double Trizol method. Isolate RNA by Trizol as usual, then air dried pellet treat with DNAse, then add another 1 ml of Trizol into DNAse/ RNa mixture and isolate RNA again w/o DNAse treatment. Also adding glycogen (see instructions for Trizol) into Trizol itself or into isopropanol helps with higher RNA yields. Make sure you never pick up interphase when collect aqueous phase after chloroform addition- better pick up less aqueous phase, then get DNA/proteins contamination. Also combination of Trizol/columns method will help a lot to get pure RNA. After addition of chloroform and collecting aqueous phase you need to add equal volume of 70% (important!, not 100%) ethanol to aqueaus phase and put mixture on RNeasy columns, then go through steps written in the kits protocol. Also if you have too many dead cells your A260/280 ratio will be much lower compare to healthy (>90%) viability cultures. So you can use Ficoll for example or dead-cell removal magnetic kits to get rid of dead cells. Good luck!
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Hello everyone! I have started a primary cell culture from murine heart, bladder and tight. I need to do a proliferation assay using EdU-Click kit, and I was wondering how long should I incubate the cells with the EdU. I optimized the protocol on AsPc-1 (pancreatic cancer) and PS-1 ( satellite cells) cell lines, and incubation of 1h was fine, as the cells replicate quite fast. But with primary cells I guess the situation is a bit different, they replicate slower, so I guess 1h is not enough. Has someone used Edu kit with primary cells? Do you have any suggestions?
Thank you in advance!
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Hello Elisabeth,
Here is an example, where primary cultures of mouse ventricular cardiomyocytes were labeled by 2 μM EdU for 12 h:
In general, it's a balance between EdU concentration and length of incubation time - higher EdU concentrations allow a shorter incubation time while lower EdU concentrations for a longer incubation time.
I suggest using a concentration of 10 µM for 1-2 hours as a starting point.
Do you want to do a pulse labelling experiment or monitor the synthesis of new DNA over a longer time?
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I need to check protein expression in primary hepatocytes after some treatment through WB analysis. I am facing problem regarding amount of protein in cell homogenate. The protein concentration is coming very less (Approx. 1 microgram/mL). Can you kindly provide me a protcol in details regarding WB analysis with primary cells? I am here sharing the method which i followed-
  • Collected approx 2 million cells in a 1.5 mL of centrifuge tube.
  • Washed twice with PBS/DBBS.
  • Added 100 micro liter of lysis buffer and kept on ice for 30 mins.
  • Vortexed vigorously.
  • Sonicated for 1 min and centrifuged at 12,000 g for 10 mins.
  • supernatant has collected to make sample for WB.
Thanks in advance.
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If properly lysed, 1mio of any cells should give you roughly ~ 100 µg, so you should get to around 1mg/ml in protein assay, not 1000 times less.
So the basics: 1: You have enough cells, e.g. you see a clear cell pellet before adding lysis buffer? 2. If lysis works, your pellet after lysis centrifugation should be much smaller than the cell pellet. 3. Protein detection: Most assays do not detect such low amounts as 1 µg/ml....(e.g. lowest for BCA is 5µg/ml) You're sure there is no calculation error and you indeed already have 1mg/ml?
Best
Christian
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Dear All
Chicken respiratory viruses such as infectious bronchitis and avian metapneumovirus isolation has been demonstrated on tracheal ring organ culture. While classifying cell culture system, organ culture in its true sense is used in plant tissue culture (root and stem culture), however, misnomer (in my opinion) has been associated with tracheal ring as organ culture instead being calling it as primary cell culture.
I need opinion of scientific community to erase this confusion
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It seems to me that it is more organ culture than primary cell culture.
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Hello everyone,
I am trying to isolate cardiomyocytes from a day old rat pups for patch clamping. I use trypsin 0.1% and 50microgram/ml DNAse 1 in HBSS to digest the ventricle pieces. Cells are isolated by 5-minute rounds of digestion for 10 times in 37degree c.
Unfortunately after 2-3 rounds of digestions the tissues pieces start to clamp together and become a blob of transparent jelly. It's very hard to break it up by triturating with a pipette. Also the cell yield is very low .
Will it help if I add EDTA to the trypsin solution? Also is coating the culture surface necessary for cardiomyocytes? I use Corning T-75 culture flasks.
Thanks in advance for your kind response.
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Hi everybody,
I'm treating my contaminated cell cultures by primocin. What experiences do you have? What is the optimal length of treatment to obtain healthy cell culture? Do you use primocin all the time as preventive step or only in the case of contamination? 
Thanks a lot
Pavla
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Routine use of antibiotics in cell culture is not recommended. since , antibiotic resistant strains may develop and may cause resistant cryptic infections such as mycoplasma. Moreover, some antibiotics may have effects in cellular functions. Otherwise, primocin is a good choice.
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Hi, I am culturing cortical neural cultures derived from P0-1 rats. Cells are plated onto poly-D-lysine coated dishes and survive nicely in NeurobasalA + B27 + GlutaMax + PenStrep - 50% of the media is replaced with fresh medium every 3-4 days.
I use the cells between DIV19-21. I am looking at the extracellular vesicles (EVs) they secrete and therefore before adding treatments I have to do a full media change in order to remove the endogenous EVs - I remove exisiting media, quickly but gently add fresh, warmed media containing my treatment (a neuroprotective peptide) and return to the incubator for 24 h. Culture dishes were chosen for treatment because they looked beautiful and happy on the day of treatment, however, when I check them at the end of the 24 h they are clearly unhappy/dead. I have ruled out contamination of the media as the same media is used in younger cultures and does not cause cell death.
The issue seems to be associated with older cultures, e.g. DIV15+, as sometimes even the half media changes cause this issue, but prior to DIV15 the cells are not affected by media changes. Changing media less frequently after ~DIV14 has helped my cells last longer, but the media change required for the treatment itself still causes the problem.
I am wondering if others have encountered this problem. I have read papers (e.g. Driscoll et al., 1991; 1993 in Journal of Neurochemistry) suggesting that glutamine/glutamate is the cause the cell death observed after media changes - and wonder if I should be leaving GlutaMax out of the media at these later DIV. Alternatively, could the PenStrep be a problem at these later DIV? I have read that some only add it in the early days of culturing and then stop including it later on. The Driscoll paper suggested that PenStrep actually exacerbated the problem. Any advice or suggestions would be appreciated!
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We had similar problems using mouse embryonic neuron cultures. When aged, the cells do not appreciate to be dry (without medium) even for very short periods. Before treating those cells we change our medium by sequentially removing very gently (without any swirl) half of the volume contained in the well. After three or four operations we consider that the medium is now renewed and we add the substance to test, keeping one well as control. We prefer also glutamine to glutamax which appears to be toxic in some cases.
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Hi,
As harvesting spheroid from primary cells takes longer than cell line and as hanging drop method is best method among others is best for spheroid formation, the changing medium would be an serious issue. if someone used hanging drop for producing spheroids, can someone explain me how and after how many days the medium was changed for their spheroids?
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Do we still use such methods? It is too old method. Nowadays, you just have to use ultra low attachment flasks or nunclon sphera flaks and you can easily get sphere.
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Hi everyone,
I'm beginning to work with brain tissue, and I will have to use FACS to isolate neurons from a brain tissue sampling.
I have frozen at -80°C pieces of brain tissue (0.5cm3 diameter, both cortex and white matter) in DMEM+FCS+DMSO.
Now I have to dissociate the tissue to FACS: do you suggest a good commercial kit for dissociation, to obtain a good number of viable neurons?
Thanks a lot, and if you have any other suggestion it is appreciated as I'm new in the field!
Have a nice day,
Sara
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Hi, can the Adult brain dissociation kit be adapted for other animals. My interest is for macaque species
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I'm plating fibroblasts on collagen gels (type 1 rat tail collagen, 1 mg/mL) but I have found that when I need to separate my cells from the gels, collagenase is too harsh and results in a lot of cell death. Is there any other way to digest collagen with low impact on the cells?
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Hello Catherine,
Don't use Collagenase. You can try to use trypsin/EDTA to separate cells from the collagen gel. I am regularly using the trypsin/EDTA solution. It works perfectly well.
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My experiment is isolating primary cells from biopsy of different patients.
And then to culture the primary cells in two conditions, one is the negative control, and the experimental set-up is to culture with more hormone. I had cultured them in parallel (both are freshly cultured immediately after I isolated them from biopsy).
Then I got the assay result in two different culture conditions, I considered my results are paired result of each patient, so I performed paired T-test.
But when I presented it to my colleagues, she said paired sample t-test may not be appropriate here because there is some strict regulations about using paired sample t-test with samples from cell culture. I had briefly search for it for a while but cannot find it.
Can anyone tell me whether there is such regulation?
I had also performed independent sample t-test with my result, and got a much larger p-value. I think it is the individual difference between patients generated larger variance in the analysis.
Could anyone tell me the best test for my data?
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If you harvest during exponential growth clearly that may play a part in your interpretation of experimental results. However that was not the issue. All cells were of the same cell type to within a small number of spontaneous mutations perhaps. The only difference between the plates is that one plate contains hormone while the other does not. The inoculate for each paired plate comes from the same individual. I assume growth curves were obtained for cells with and without hormone so you know if hormone has an effect on cell growth and what if any that effect is so if such an effect exists you may consider it with the rest of the experimental results. Given all this unless some strange effect happens that requires explanation. This is statistically a paired t test. Any effect on the growth is known so any correction necessary can be made, such as was suggested by Dr. Ebert. Again when all necessary corrections, transformations and what have you are finished. The comparison is the paired t-test. This is exactly how we did chemical mutagenisis studies in Herman E. Brockman's lab many years ago. See the link for publications from some in the group with Materials and Methods sections. I apologize for my poor description but this was 50 years ago for me. Mea Culpa.
Again, I see no reason why a poorly remembered something or other affects either the statistics or the biology. As always the assumptions of the test should be checked. Best, David Booth
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Hello,
I am isolating primary murine glial cells from different neonatal knockout strains (p0-p2). After several rounds (up to 4 times) of harvesting the microglia, I am dissociating the astrocyte monolayer from PDL coated 75 cm² flasks (containing 2 brains). At this step, I am having issues getting single cells, the astrocytes are sticking together and forming big chunks of cells.
Although I have aleady tried different methods of dissociation such as using different kinds of narrow pipettes, trypsine, DNAse, papain, cell strainers and so on, I am still getting big chunks of cells. The cells seem to be fine since live/dead staining with trypan blue indicates that the cells are pretty viable (less than 5% dead cells).
Does anyone have a clue what makes the astrocytes stick together after their dissocation from the cell flasks?
Thanks in advance!
Kind regards,
Laura
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Hi Laura, I found an interesting paper tht compares different approaches for isolating cells from brain tissue, maybe it can help you. You might want to try change your reagents.
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Hi, I had primary cell culture on trigeminal ganglion cell and I have to detach these cells.
So I'm planning to use TrypLE Express or just trypsine, but I wonder what reagent would be suitable for neuron protection.
I only had experience on cancer cell line detachment using trypsine, and I'm not sure there are any difference on neuron cells.
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John V. Stokes
Thank you John! I will look for UpCell dishes. Have a good day
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Hi 
I want to sort fluorescent labelled cardiomyocytes with FACS and collect them and do RNA-seq on them. I am using a lysis solution containing protease and collagenase after mincing the heart but I am a bit worried about the RNA-ases that may start working and degrade the RNA altering my seq. Do you have any protocol or suggestion on how to stop such a change or potential change in RNA profile in cardiomyocytes? Do you think RNA Later may help circumvent it?
Thanks a lot for your suggestion!! Appreciate it.
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I realise this question was posed and answered some time ago, but I would be interested on hearing some more on the topic - would anyone suggest adding RNAse inhibitor prior to sorting during cell isolation, or is it sufficient to add it after sorting the cells?
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Hello, I am trying to generate primary cell cultures from different tissues of adult zebrafish (including gills, skin and muscle). I have tried several methods for cleaning the whole fish (hypochlorite, ethanol) and the tissues pieces after dissection and before digestion. Nevertheless, after plating I can see not only cells but also bacteria. I have tried different antibiotic combinations including penicillin/streptomycin, gentamicin and primocin but I still get a high amount of bacterial contamination. Any tips on how to improve this? is this normal?
P.S- No many publications mention the contamination of primary cultures with the own fish's bacteria. I have only found this one from 1976 - THE USE OF ANTIBIOTICS IN THE PREPARATION OF AMPHIBIAN CELL CUL TURES FROM HIGHLY CONTAMINATED MATERIAL,
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The majority of bacteria in fish are in the external mucous coat or the gut. So one approach is to minimize these microbial populations by changing fish husbandry prior to collection:
  • 1-2 days before collection, stop feeding fish.
  • Place fish in dedicated tank of sterile aquarium water supplemented with broad-spectrum antibiotics.
  • Fish density should be very low (much lower than usual) and the water should be well-agitated with bubblers.
  • Once anaesthetized, rinse fish again in sterile system water, and briefly spray/wipe with a surface decontaminant such as 70% ethanol, topical hydrogen peroxide, or diluted iodine.
  • Dissect the fish in a pre-cleaned surgical area such as a UV-sterilized containment hood, an autoclaved dissecting pan, or a clean bench covered with a sterile drape.
  • Autoclaved or flame-sterilized instruments are advisable.
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Hi!
I am trying to select the lowest amount of blasticidin needed for selection in murine primary neurons. I have tried concentrations from 1-8 ug/ml and had no difference in the fitness between the cells. The cells did appear dead, the axons were disintegrating but the cells didnt actually detach from the wells.
I am not sure what to make out of this, could I use blasticidin for selection and passage the cells & count on that the dead cells wont attach but that the live ones will?
Any input would be appreciated.
Best,
Linn
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Hi Prasanth and Andreas,
I will provide more info on the experimental setting. So I am working on wild type mice and the neurons are first isolated and plated. Afterwards I am aiming to transfect them with 1) pX330 plasmids expressing cas9 and the gRNA for my specific target and 2) a selection vector harboring the sequence that the gRNA should recognize and cut. It also contains a frameshifted non-functional blasticidin resistance gene. When the cas9 cuts the sequence on the plasmid a frameshift will repair the blasticidin gene.
Before starting the experiment I want to find o