Science method
Primary Cell Culture - Science method
Explore the latest questions and answers in Primary Cell Culture, and find Primary Cell Culture experts.
Questions related to Primary Cell Culture
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.
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.
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.
Hi Everyone,
Should I add L-glutamine during media preparation if the culture media (RPMI 1640 with l-glutamine) already contain the same?
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.
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?
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,
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?
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?
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!
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....
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
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!
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.
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?
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?
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.
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!
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.
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
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!
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?
what kind of media and how to culture it?
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?
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.
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 :)
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).
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.
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!
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!
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!
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?
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!
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
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.
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
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:
- I use P1-P2 rats instead of embryos
- I coat coverslips with Matrigel and culture the cells in 6-well plates
- I added a collagenase IA digestion step (60 minutes) to get rid of the capsule and I digest with trypsin only for 15 minutes.
- 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
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
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!
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.
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?
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
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?
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
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!
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.
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?
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!
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.
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.
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
Is it a problem, if I keep them overnight and replace with growth media next day?
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?
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!
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.
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
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.
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
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!
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?
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
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?
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?
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
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.
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.
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,
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