Primary Cell Culture - Science method
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Questions related to Primary Cell Culture
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!
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.
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.
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!
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?
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!!
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
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?
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?)
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.
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?
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.
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.
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?
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?
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.
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....
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
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!
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 ?
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.
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?
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?
I have been trying to culture primary fibroblast cells from mouse skin for awhile now according to the following protocol:
- 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?
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 :)
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!
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?
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!
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.
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
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.
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?
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?
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
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!
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).
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.
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?
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.
Is it a problem, if I keep them overnight and replace with growth media next day?