Science method

Primary Cell Culture - Science method

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Hello, I am wondering if there is a good way to normalize my data. For background, I collect conditioned media from primary cells and then perform radioimmunoassay on them to determine the concentration of progesterone secreted into the media. I know RIA isn't used these days very much, but theoretically it is very similar to ELISA. There does seem to be differences between my treatments and controls, but without normalizing I'm not sure whether it is due to my treatment or something else. Total protein would probably be best to normalize to I imagine, but I do not have this as we only extract for RNA after the treatment period. I have been wracking my brain to think of some way to normalize the data such as normalizing to the % increase/decrease between the treated and controls but not sure if this would make sense. Any help would be appreciated!
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Yes, you can normalize your progesterone data to a housekeeping gene using qPCR. Here's how you can do it:
  1. Run a qPCR assay for both your housekeeping gene (e.g., GAPDH) and your target gene (progesterone) on your cDNA samples. This will give you the Ct (cycle threshold) values for both genes.
  2. Calculate the ΔCt (delta Ct) for each sample by subtracting the Ct value of the housekeeping gene from the Ct value of the target gene. This ΔCt represents the relative expression level of your target gene normalized to the housekeeping gene.
  3. If you have multiple samples in different treatment groups, calculate the ΔΔCt (delta-delta Ct) by subtracting the ΔCt of your control group from the ΔCt of your treated group for each sample. This gives you a measure of the fold change in gene expression between the treated and control groups.
  4. To normalize your progesterone data, you can use the ΔΔCt values as a factor. Divide the progesterone concentration of each sample by the corresponding ΔΔCt value. This will adjust your progesterone data relative to the gene expression changes observed in your qPCR experiment.
Regarding combining methods like housekeeping gene normalization and percentage change:
  • If you want to combine housekeeping gene normalization with percentage change normalization, you would first normalize your progesterone data to the housekeeping gene as described above.
  • Then, if you want to account for percentage changes, you can further divide the normalized progesterone values by the percentage change factor. This would mean that you're adjusting your progesterone data for both gene expression changes (using the housekeeping gene normalization) and percentage changes between treated and control groups.
  • Essentially, you'd have two normalization steps: first to the housekeeping gene and then to the percentage change factor.
All the best
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Urgent question! Do human cells survive at -20 degrees for couple of weeks or do we have to be store them at -80 or below. Any suggestion will be greatly appreciated?
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If you are going to culture these human cells in future (after a few weeks), then you may have to store them at -80 degree C or in liquid nitrogen but in a freezing medium containing 90% complete culture medium + 10% DMSO. You will also have to freeze them gradually at -1°C/minute using Mr.Frosty container. This container is designed to achieve a rate of cooling very close to -1°C/minute, the optimal rate for cell preservation.
Human cells will not survive at -20-degree C.
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I did a primary culture with the objective of isolate hepatocytes, but I only obtained these cells. I need a bit of help trying to identify what kind of cells are?
I took a few photos of them.
I would really appreciate help with identifying these kind of cells.
I believe that I took the photo of a fibroblast and a couple of picture of death cells but I am not sure.
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Top: a - looks like fibroblasts or neurons/glia. b - looks like fibroblast. c - looks like non-adherents (e.g sphere f)or dead cells. d - looks like a small sheer or dead cell cluster
bottom: a - looks like some sort of epithelial cluster. b - looks like cells infected with bacteria
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Hi, everyone
Does anyone know how to isolate bladder smooth muscle cells without getting other cells like interstitial cells or fibroblast cells for primary cell culture ? Also, any suggestion on which media is better for the growth of mouse smooth muscle cells.
Thank you.
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Thank you Dr. Saif
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I need to do a primary culture of Non parenquimatic liver cells from mice and althought, I have the protocol for the obtaining and isolation of these cells, I do not know which medium to use, what porcentage of FBS use and what and how much supplements use (like Glutamine, antibiotics, etc).
I would really appreciate the help!
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To perform a primary culture of non-parenchymal liver cells from mice, it is essential to choose the appropriate medium, determine the percentage of fetal bovine serum (FBS) to use, and decide on the necessary supplements. It is important to note that specific protocols may vary based on the intended application and the preferences of your laboratory.
For the medium, a commonly used choice for primary cell cultures is Dulbecco's Modified Eagle Medium (DMEM) or RPMI-1640. Both media are widely available and suitable for the growth of liver cells. The selection of the medium may depend on the specific requirements of your experiment or the protocols followed by your research group.
Regarding the percentage of FBS, a common range is between 5% to 10%. The choice of the exact percentage depends on the specific cell type and experimental conditions. It is advisable to optimize the FBS concentration based on the viability and growth characteristics of the non-parenchymal liver cells in your particular experiment.
As for supplements, commonly added components include L-glutamine, penicillin-streptomycin (antibiotics), and non-essential amino acids. The recommended concentration of L-glutamine is typically 2 mM, while the antibiotics are generally added at concentrations of 100 units/mL of penicillin and 100 μg/mL of streptomycin. Non-essential amino acids are often added at a final concentration of 1% or as specified by the supplier.
Additionally, considering the variability in experimental conditions, it is recommended to perform optimization experiments to ensure the optimal culture conditions for your non-parenchymal liver cells.
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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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Hi..Rather than establishing the resistance cell line,I've found some published articles compared the resistance of various cell lines (from the same cancer type) following drug treatment. Cells with greater survival rate following drug treatment were selected as a resistance model
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Any suggestion, recommendations, things I must know. Thankyou very much.
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Additionally, I recommend thoroughly cleaning the bones in PBS (i.e. removing as much tissue as possible- I use a scalpel while the mouse is pinned for resistance), and then flushing the bones in fresh PBS.
After culturing with L929 enriched media, we make sure they're macrophages via flow cytometry.
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I'm using Mitotracker (Green) and MitoSox (Red) probes to study ROS generation in oligodendrocytes from primary cell culture. I wonder to know if these probes could be incubate at the same time or they should be incubate sequentially. Thanks!!
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Yes, you can use both the dyes together. However incubation time may differ for both. For me incubation of 15 mins is sufficient for MitoTracker staining, however MitoSOX needs 45mins of staining.
You can use MitoTracker with MFN-1, DRP-1 or other mitochondrial dynamics marker to asses mitochondrial fission or fusion.
Hope it helps.
Thanks
Samir
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Hi,
I am culturing primary Motoneuron from mouse spinal cord on Porn and laminin coated coverslips. By day 7 I get about 10% survival of the neurons. The seeding density was 5000 cells. What can I do to increase the survival of the Motoneurons.
please share your thoughts
thank you
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Hello Sonia, could you provide more information? Are you using embryonic or neonatal mouse/rat? Are you following a published protocol? White size of coverslips are you using or density of cells per mm2? What media with/without supplement? Just by the look of it it sure looks like the density of the culture is too low, but it's hard to say without the details.
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I did DRG primary cell culture. We did good counting with poly dl and laminin but most of the cells are not attached and make clumbes. I must say that I did not rush to then to ging to clumb.
what do you think is the problem?
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Rabeah Al-Temaimi, I don't think the laminin would be a problem cuz I saw different protocols that used laminin and they do not have any problem. actually, my problem is that my cells didn't attach so I do not have even fibroblast in the plate.
But I think the problem could be with the coating.
Thanks for your answer.
Best regards,
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I am trying to grow human cardiomyocytes from primary cell culture for the first time and wanted to check how ImageJ worked for confluency. However, I don't have access to phase contrast microscope so need to make do with a compound microscope for now. Anyone has any suggestions for plugins / add ons or if there is any other alternate for counting confluency which isn't manual (to keep results consistent enough?)
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Hi Aarohi Shah. ImageJ works with any digital image, so images acquired from any microscope can be analyzed. If contrast is good, you can threshold your image and use Analyze Particles to measure the area fraction of cells in your images.
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Hi all,
I'm growing primary basal cells from lung digests, and was wondering if anyone has recommendations for cell freezing media for primary lung basal cells.
Previously I have been using Cryostor CS10, however I'm finding quite costly and would like to make my own.
My expansion media is Pneumacult EX Plus.
Many thanks,
Sam
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My pleasure! Let us know how your cells recover from freeze-thaw and how it compares to the current freezing medium!
Cheers,
Martine
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24h? 48h? 72h?
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After placental delivery, the umbilical cord is stored in PBS at 4 degree C. You should isolate HUVECs preferably within 12 hours, but always within 24 hours after placental delivery.
Best.
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Hi everyone,
I isolate tenocytes from mice tendons. I extract the mice tendons, place them in collagenase overnight and then seed them on T75 flasks. I have been using the same medium as the people before me (DMEM42430-025 +10%FBS,+1%P/s+1%NEAA).
The cells were already looking strange in the previous isolation so I seeded them on collagen coated flasks this time. They still seem to differentiate into some kind of weird cells.
I was wondering if anyone has any experience working with primary cells and have observed this before?
Thanks!
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They are OK. Do not worry.
If you want another method, where you culture small pieces of tendon in culture, and fibroblast proliferate, either from human or mouse tendon, it worked for me. This procedure can take 10 days for fibroblast to come out of tissue pieces. But they are pure fibroblast and no enzyme used. Here are some reprints. Once you get monolayer, then you can expand normally.
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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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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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I want to overexpress a mouse protein into mouse peritoneal macrophage, so which method will work best viral transduction or plamid transfection and if anybody has protocol for the same please let me know.
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Hey Juan,
we struggled for a long time to transfect our macrophages. We had even more trouble because we use ex vivo peritoneal macrophages. They are very delicate and react nearly to everything. Viromer substances or Lipofectamine either did not work or killed the macrophages. RAW cells are easier to transfect (cancer-like cell line). However, we recently established a method for transfection with the JetMessenger transfection reagent and in vitro transcribed and immunologically silenced mRNA. Modifications during mRNA transcribtion were:
Removing any traces of DNA templates (because this induces macrophage death).
Use of 5-methyl-CTP and pseudo-UTP, which are bases that reduce immunogenicity and enhance stability of the mRNA.
Dephosphorylation of the 5’-ends of the in vitro transcribed mRNA by Antarctic phosphatase to prevent recognition by the RIG-I complex.
All of these steps together led finally to sucessful macrophage transfection, but it took long to get it right. For a detailed protocol please have a look at:
Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA. J. Vis. Exp. (Pending Publication), e60143, In-press (2019).
It worked for peritoneal macrophages, BMDM and MEFS.
But it only delivers mRNA and not plasmids. Tissues macropages go instantly into apoptosis, when we tried to transfect DNA.
I hope this was helpful to you.
All the best and a happy new year,
Marc
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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!