Questions related to Fibroblast
Hi, I'm working on oxidative stress of fibroblast and I want to induce stress on cells as a control. I have heard about induce strees with H2O2 but I'm not sure about concentrations. Could someone tell me what is the concentration they use to induce stress? or another method for induce stress on fibroblast?
I am trying to isolate keratinocytes from skin biopsies for single cell sequencing. I would like to have just keratinocytes without any immune cells or fibroblasts. Can anyone suggest a method? I am alreading inclined towards using dispase to separate the epidermis from the dermis, trypsin-EDTA to digest the epidermis and MACs to sort out CD45 positive cells. My worry is that my cells will not be viable afterwards.
Our primary cells are not proliferating and the cell culture has an aberrant debris-like phenotype. Has anyone encountered such a problem?
Mycoplasma test is negative.
Appreciate your help
Wondering if TrypLE Express cell dissociation agent is significantly better than other options for culturing mammalian cell lines. Currently mainly using Accutase (for fibroblasts and epithelial cells, NOT stem cells) and sometimes also trypsin for specific cell lines. Any positive/negative experience with TrypLE Express enzyme? Main attraction is that it does not require neutralization as trypsin does, but has longer incubation times. Would appreciate any comments and suggestions.
My study needs to test the adipose tissue derived stem cells' effect on the fibroblasts. I planned to perform a indirect co-culture of these two kinds of cell through seeding ADSCs in the 0.4 um transwell and seeding fibroblasts in 6-well plate. The question appears because ADSC grows slower than fibroblast, so I need to wait ADSC until it reach 80% confluence. While, during ADSC culture in transwell, I found that culture medium leaked to the well under the transwell. So should I add medium in the well also? And if the medium didn't leak into the well, will the secreted cytokine or exosomes from ADSC transport to the medium when co-culture with fibroblasts?
I am culturing human primary keratinocytes and fibroblast together to make an organotypic model of skin. I already had the following collagen solution in my lab so I used it.
Unfortunately, this solution does not transform into a 3D gel when I add NaOH in it.
If anyone has a similar experience then kindly guide me.
is there any possibility that these cells would make a differentiated skin model without a solid collagen layer below them?
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?
These are rat dermal fibroblasts that I've been growing for the past 2 days. However, they're not reaching confluence at all, even after I've changed the media. I make sure that the media (DMEM + 10% FBS) is warm and sterile; however I still see they're not growing. A lot of the cells are also dying. Could anyone suggest next steps?
I thaw an old cryotube of L929 immortalized mouse fibroblasts with very low cell viability (less than 5%) and they seem to be contaminated by huge multinucleated cells (see images). I am not going to use it for an experiment, but I would like to know what could it be in the case I come across this kind of cell formation in the future.
I wanted to investigate the inflammatory response of the lipopolysaccharide from the bacterium Porphyromonas gingivalis (P.G.) on human gingival fibroblast cells. To try it out, I wanted to measure the expression level of the proinflammatory cytokine IL-8 with ELISA. As a result of my research, I treated the cells with P.G. lps at different time intervals (1, 3, 6, 9, 12, 24 h) and at a concentration of 0.1, 1, 5, 10 and 50 µg/ml. However, I could not observe a color change according to the standard in any way. What could be the reason for this?
Suddenly, we all have in our lab multiple beta-actin bands.
To check if this is because of the antibody, I used an old sample (a month old) together with newer samples (fresh and also one thawed after being frozen for a week). Interestingly, the multiple bands only show in the fresh/new samples. Is there something wrong with the cells in the fresh samples and are these bands reflecting actin degradation?
Other people in the lab have the same thing. We see it both with fibroblast and iPSC-derived neuron samples. We didn't change anything in our protocols, we all performed the experiments separately and it has been going on for almost a month now.
Does anyone have experienced this as well?
I included a western blot, showing three bands in the new samples and only one in the old sample.
I transfected my cells (primary human dermal fibroblasts) with a plasmid containing the gene of interest and a C-terminal turboGFP tag.
I'm able to see the turboGFP fluorescence using a fluorescence microscope.
I fixed these cells in ice-cold methanol and paraformaldehyde.
However, when I performed an immunofluorescence, the green fluorescence of the turboGFP almost completely disappeared.
How can I fix and permeabilize the transfected cells preserving the fluorescence of GFP and of secondary antibody?
Could you give me a good protocol?
I am trying to isolate enteric fibroblasts from the small intestine and colon of mice. I am successful in isolating fibroblasts from the colon, but not from the small intestine following the same protocol. I do the following:
1) Take the gut and clean with PBS
2) Keep the gut in DMEM with NEAA, glutamine, penicillin-streptomycin, gentamicin, or in MEM-Hepes with penicillin-streptomycin until the tissue is processed (in ice)
3) Fragment the organ as thin as possible with a scalpel in the DMEM recipe mentioned above.
4) Add 1 mg/mL collagenase, 1 mg/mL dispase, and 10 µg/mL DNase I
5) Digest the tissue at 37°C for 90min, 65rpm. (I have tried 30 and 60min as well and failed)
6) Filter through a 70µm diameter cell strainer
7) Centrifuge 300g, 5min and remove the supernatant
8) Resuspend the cells and culture in DMEM (recipe as above) supplemented with 10% FCS.
* I am not using collagen-coated plates and the colonic fibroblasts grow well there...
Thank you for your help!
I'm currently working on decellularizing ECM produced by fibroblasts to be used as base for cancer cells culture. The problem is that even with the gentlest wash the dECM still comes off the surface easily and result in a solution containing broken dECM. I'm thinking that for the purpose of my research I can also centrifuge the solution, collect that broken dECM and embed it into hydrogel for use. Does anyone has similar experience and know if it's feasible and if so what would be a good centrifuge setting to use? Or if you have better tips on how to make dECM stay more firmly on a well plate surface you're welcomed! Thanks
I am trying to find a cheaper replacement for the item below. I've found a few options but i'm confused why some say 154 aa or 147 aa. I understand that the aa stands for amino acids but will this make a difference for what we are trying to use it for. We need it to supplement our cells that we are reprogramming from fibroblast to iPSCs.
I am currently trying to grow out primary ectocervix epithelial cells from patient tissue samples. I firstly started with the tissue out growth method where I allowed the cells to grow out from the ectocervix tissue piece. When I have a monolayer of cells I trypsinize the cells and passage. After trypsinization, the fibroblast-like cells attach to the plate however, the epithelial cells do not re-attach and end up dying. I have now tried the Dispase II method and after digestion of the tissue followed by trypsinization the cells once again don't attach to the plate and end up dying. The only success I have had is with the patient derived fibroblast however, the epithelial cells do not even survive one round of trypsinization. Does anyone have have any suggestions on how to overcome this problem?
Hello, I am learning how to grow cells and I have been having trouble seeding primary dermal fibroblasts in a 6-well plate. After seeding the cells grow in the edges of the wells but not in the center. If anyone is kind enough I would love to hear what could be causing this? I have attached a few pictures (taken from the center to the edge of the well) that shows the issue. Thank you!
I am looking for the number of fibroblast in healthy human skin- dermal fibroblasts and I am not dividing them into subpopulations.
Does anyone have a good reference for this?
Optimal would be a value in [cells/cm^2 skin]. Maybe from histological countings or from isolations- How many fibroblasts can be isolated per cm^2?
It would be awesome if someone had an answer :-)
Hello to all
I observed a strange phenomenon with CHO-K1 following the change of serum batch (it is surely the toxicity of this batch but I am very curious about the possible nature of this phenomenon. In practice, I will simply change serum. But the question remains open)
At the beginning of the culture, CHO-K1 grow normally. I observe a normal triangular morphology (round to cubic). The growth is fast as usual. But, once at confluence and not before, they start to take the shape of very elongated cells in parallel arrays evolving like fibroblasts.
I tried to dilute them as much as possible at the beginning of the culture. No change. They take a normal amount of time to grow to confluence, then they become fibroblasts-like.
Has anyone seen this kind of phenomenon? It is probably toxicity in the serum, but usually the toxicity is greater if you dilute the cells (" dilution death "). This is not the case this time. Any ideas would be helpful.
My colleagues and I work with primary mouse embryonic fibroblasts (pMEFs) for two years all was going well. Beginning of 2021 we noticed are cells became very delicate, often detached and died within 1 week. We had just moved our tissue culture space, so we went back to the old incubator, and this seemed to fix the problem (There was something off with the new incubator). Around 8 months ago we noticed our cells becoming very temperamental again. Being ok in T75 flask then not surviving passed a week after plating for an experiment. This time we noticed strange morphology of the cells (see attached images), almost like the cells where crystallising. Then the cells started doing this all the time. We have 4 different genotypes, and each genotype is affected to a similar degree. We have changed incubators to a third one and the cells seem slightly happier, but some cell lines are fine with others having the strange crystal-ish morphology. It seems to be completely random which cells seem ok and which go bananas. This happens with both reived cells (from liquid nitrogen) or from freshly harvested embryos.
We have tried different incubators, cleaned out/autoclaved incubators before use, freshly made media*, freshly made media with brand new bottles of EVERYTHING (in case of some lot issues), different users (ie I revived them, my colleague dose the first passage and vice versa)
We are at a complete loss as to what might be going on with our cells and I am looking for any advice or hints at what might be going on.
* Tissue Culture solutions we use
DMEM low glucose – Sigma D5546, kept 4C
Pen/Strep – sigma P4333, aliquot and store at -20C
Trypsin/EDTA 0.25% - Sigma T4049 once open keep at 4C (or aliquot and store -20C)
L-glutamine 200mM – Sigma G7513, aliquot and store at -20C
FBS (heat inactivated) – Fisher (Gibco) 111543407 (100ml) aliquot and store at -20C (try Sigma/Merck F9665-100ml £16.25 quote HLM <31/10/19)
PBS – 100ml made up from tablets (Oxoid, stores), MQH2O then autoclaved, store at 4C
Complete Media (cMem; kept at 4C) = DMEM (500ml),
FBS (50ml, final conc 10%),
Pen/Strep (5 ml, final conc 1%),
L-Glutamine (2.5ml, final conc 1mM, 0.5% v/v)
We have not changed brand or even suppliers of these components from when our cells were behaving nicely.
Notes from colleague
Tissue culture issues
pMEF, 8 embryos, 0.70 TC 5% CO2 37’C. low gluc DMEM made 14.1.22, 31.1.22. Struggled once in T75 – future, keep some in t25 as long as poss…
end Jan, clean/autoclave 1.38 10% CO2 37’C, re-calibrated.
pMEF wt xiao, thaw from LqN2, struggled after first split.
Mid jan – issues with LqN2 storage, left to go empty – resolved Feb, will be topped up every Monday and checked.
pMEF 1 embryo wt. 0.70 TC 5% 37’C, all ok, frozen down. cMEM 14.1.22. all seemed ok
pMEF xiao that from LqN2, also pMEF02/03 from LqN2. Revive ok, struggle after first split, initially cMEM made 12.1.22 with old L-glut? Made fresh cMEM 14.1.22. Plated all geno 96w and 24w for diff from xiao cells (old diffMEM in 4s1.38 cloudy, orange), by day 5 ALL cells clumpy, change pH, unhappy
1.38 incubator clean/autoclave recalibrate over Xmas break.
Thaw pMEF xiao, 0.70 TC 5% CO2 37’C. Struggle after first split and plating, start diff day 5+ diff’ing cells clumpy, this is diffMEM that went off
Mid Nov – 4S1.38 3t3-BioID, thaw by Rachel – from -80 or LqN2, subculture ok, frozen down ok.
pMeF xiao, 4S 0.70 5% 37’C. all geno, struggle after first split. Thaw more 08.11.21, plated but slow growing, diff cells in 1.38 10% TC all ok, diff nicely to day8?. 5% TC in 0.70 plated cells not growing as expected.
Thaw pMEF-x for RNA Seq from LqN2 to 0.70 TC 5% CO2 37’C, all generally fine, plated on 2nd passage or greater – 12w, 24w, 96w, + RNA. Grow fine for RNA, flasks ok. These cells seem to sub cult ok. Diff pMEF-x wt with new diffMEM (181021) compared to old diffMEM (031021) with either new diff reagents or old diff reagents (dex, biotin (rest ‘fresh’ anyway). All diff cells ok at day 8 and nicely diff’d whether old/old, old/new, new/old, new/new.
Thaw pMEF-x wt from LqN2 (p5), plated at p6, grow fine (0.70 TC 5% CO2 37’C). To check diffMEM old vs new. Undiff = fine; diff + old = floating, clumpy etc; diff+ new = fine at DIC2, all clumpy by day 7.
Thaw 3t3 parental line from LqN2 (1.38 TC 10% CO2 37’C) to check diffMEM etc, slow growth (was frozen as 1 of 4), diff with fresh vs old media. Undiff = fine; diff + old = floating, clumpy etc; diff+ new = fine at DIC2, all clumpy by day 7
Thaw pMEF xiao, pMEF06 from LqN2. Mixed recovery, plated 96w, 24w. flasks struggle after splits. Diff +/- rapa (new stocks). Diff cells clumpy/dying day 4 (96w)
pMEF-x diff started by RJ – is this the start of diff issues? See floating cells, pale media, cloudy in diff-ing cells, undiff generally as expected. Took RNA, nanodrop  in diff<undiff (at least dec 2 fold. not seen this diff previously)
pMEF-x thaw from LqN2, 0.70 TC 5% CO2, 37’C. plated at next split (for RNA +/- diff). diff went fine, RNA15.
Cells plated mid May 21 (3t3, pMEF) seems to have gone fine, RNA for Grb/Dlk timepoints.
Now, I am isolating colonic fibroblasts from C57BL/6 mice, I have tried digestion with 0.05g/ml collagenase type 1, but very few fibroblasts are isolated.
Can any research colleagues give me some advice?
I have a NIH 3T3 mouse fibroblast cell line and a pLL 3.7 lentiviral vector. The vector has GFP (with CMV promoter) and shRNA (with U6 promoter). Upon infecting the 3T3 cell line, the cells expressed GFP. But with time and passaging, the cells are expressing very low GFP (or nothing at all). Also, the control cells (infected with vectors having only with GFP-CMV and no silencing RNA) expressed GFP initially, but very low GFP expression upon passaging. Further, western blot analysis indicates that the knockdown was not successful. It is as if the cells have found a way to silence both, the GFP expression as well as knockdown.
Any solutions to obtaining a stable, infected mouse fibroblast cell line?
I want to do Live/dead cell viability assay on my collagen gels using calcein am and ethd-1
My questions are:
a) What kind of plate for confocal microscopy should I use? I used thin glass bottom 35 mm and ibidi dishes with small well in the middle part but my collagen doesn't attach! should I change the plate or coat the plate?
b) How many cells/cm2 do you suggest? different papers use different cell numbers!
Does anyone do the same experiment or have a helpful source in mind?
I am wondering what happens to the frozen Conditioned-media (CM) that is collected from cancer cells and then incubated for 24 hours to 48 hours? Will the secreted proteins in CM such as Chemokines and cytokines be degraded? In other words, does the incubation affect the CM composition?
Dear Scientists of Researchgate,
After a lot of trial and error, we managed to grow and maintain L929 fibroblasts in-house. We managed to grow a few monolayers that looked.. okay.
So far, what worked best for me was to plate 100,000-200,000 cells in 2mL of EMEM with 10% FBS for 24-48 hours, then replace the medium layer with pure EMEM (no Fetal bovine serum added). The cells differentiated relatively well.
Our team doesn't have any prior experience working with adherent mammalian cell lines, so I was wondering - does anyone have any tips on making sure L929 cells differentiate into fibroblasts with a higher percentage? And if anyone has experience working with these cells - could you please evaluate the quality of the monolayers I have attached pictures of? Any feedback would be greatly appreciated.
I am growing human fibroblasts.
Spread 20,000 cells on a 12well plate and spread them.
When I was observing how fast the cell saturation was, the border was formed on the 5th day, so the lower part seemed to dry, twist and die. The culture medium was good enough.
Why does this happen?
I want to exposure my human primary cell line (fibroblasts) to lipids. There is not much literature available outlining how best to do this. Has anyone experience of doing so?
Specifically do I need to prepare a liposome first and if so, what do I use as the carrier/ buffer?
Thanks in advance,
Hi we recently reprogrammed fibroblasts into iPSCs, the morphology is off but we are wondering why they are still staining with tra-1-60? We picked the colonies to see if the morphology would change once re-plated but they look the same. According to this paper https://www.nature.com/articles/nbt.1580 , fully reprogramed cells stain positive for tra-1-60 but ours do not look like iPSCs.
Hi, I need to transfect mice lung fibroblasts with lenti-cre. I bought one from a company but it didn't work at all, efficiency's super low. Can anyone recommend the premade lentil-cre particles that actually work? I'm in US. Many thanks!
This is our first time reprogramming iPSCs. We reprogrammed fibroblasts using episomal vectors. Attached is a picture of reprogrammed fibroblasts at 4x on Day 19 (15 days of N2B27 +FGF and 4 days on E8). The protocol we are following mentions picking ipsc colonies at Day21+ but are these ready to be picked?
I am culturing meningeal fibroblast derived from rat P1 pups. Everytime I subculture them, they tend to attach with each other after trypsinization which makes is difficult to calculate cell viability as well as seeding density.
Any tips for the subculturing fibroblast?
I am currently using MTT assay to determine the optimal concentration of my mushroom extract and L-buthionine sulfoximine (BSO) on human fibroblast. I treated the cells with a range of concentrations of my extract or BSO for 48 hours. Following the treatment, I added 0.5 mg/mL of MTT to the cells and incubated it for 4 hours. I have noticed that some of the wells exhibit an abnormally strong purple hue compared to the other wells prior to the addition of DMSO and reading of absorbance. When observed under the microscope, the cells seem to be dead (detached and loss of spindle shape), however, a purple hue can still be observed.
I wish to know what may be the cause of this so that I may avoid it in the future. Thank you.
We are using the Neon Transfection system with Epi5 Vectors. The fibroblasts will be at passage 3 when transfected and they are from an adult donor. After transfection they will be placed in N2B27+100bFGF for 15 days and then switch to Tesr-E8. What would be the optimal seeding density for each well of a 6 well plate? Additionally is 1650V appropriate to transfect adult fibroblasts? The protocol on the website is optimized for fetal fibroblasts.
We have fibroblasts isolated from hepatocellular carcinoma tissue samples. We are planning transcriptomics. However, I am not sure if it is doable since there are only 2500-3000 cells. Which method do you advise? Might Nanostring work?
Thanks in advance for your time and consideration.
Hi, I am trying to get the dermal human fibroblasts (isolated from skin biopsies) to secrete collagen and I want to harvest this collagen and look at them under AFM.
I was wondering if anyone has a working protocol to share with me.
I appreciate your help,
I am super confused right now about the seeding density for fibroblast. I searched online and most of the company protocol said 4000/cm2. However, based on my own experience, that's clearly not the case. I then did a little bit more research and see people suggesting 10000-30000/cm2. Could anyone please explain these to me? (why ATCC for example suggest 4000/cm2?)
The aim of my study is to cultivate different types of macrophages, and to put them in co-culture with fibroblasts. I already arrived to differentiate monocytes in M1 and M2a macrophages. But my problem is that, when I put macrophages on my fibroblasts, I don't see any effect. Maybe it's because my macrophages are not activated ? What should I use to activate and to make them synthesize cytokines ?
We need to isolate cancer associated fibroblast from colorectal tissue and distant normal colon tissue. For this propose, we obtained specimen by surgery from a CRC patients. Our method is explant culture.
The obtained tissue pieces washed 3 times in PBS+ p/s and then split into multiple pieces using a fresh scalpel and attached on the surface of 6wells. After 30min, we added 2ml DMEM containing 15% FBS and p/s.
Unfortunately, no CAFs has been obtained from tissue so far.
It is noteworthy that the obtained tissues are very loose and in the first days of explant culture a large number of cells emerged from tissue, but none of them is CAFs and after a few day suspention, they died.
Has anyone had experience with CRC tissue?
I transfected mice fibroblasts with GFP construction from Addgene with a resistant gene for puromycine, I would like to understand the mechanism through which GFP integrate to mitochondria and tag it? Would be happy if someone send me some references to read about it to better understand.......
Hello, I have a question while experimenting, so I have a question.
As a porous scaffold, fibroblasts were cultured using two products, Merck PS scaffold and Lenabio SeedEZ, and cytokine assay was performed with cell supernatant.
In the 2D culture system, there is no cytokine secretion even if the incubation time is long,
In the 3D culture system, the cytokine also increased according to the incubation time.
It seems to receive a lot of stress in 3D compared to 2D, but it is questionable whether it is basically this high even without irritating substances.
Even if it is not the corresponding scaffold, is there any researcher who has done a cytokine assay after incubation using this porous scaffold?
I'd love to hear what you think.
I am using DMEM from gibco for the culturing, but cells seems not happy. I have also tried DMEM Glutamax but in all case cells do not survive more than one month. And the growth is very slow. Is anybody here has any experience in these cells? Please do me help to overcome the problem.
I am attempting to deliver a plasmid ~9000 bp in size into hard-to-transfect lung fibroblasts. Previous experiments were successful, but had extremely low efficiency, leading to longer wait time to re-grow surviving cells to confluence before proceeding to the next steps.
I've read that reverse-transfection, in which the DNA-lipid complex is deposited into the well before the cells, can increase transfection efficiency in these types of situations. Our lab uses Lipofectamine 3000 for transfections, but the only protocols for reverse transfection I can find on ThermoFisher scientific's website deal with their RNAiMax reagent (https://www.thermofisher.com/us/en/home/references/protocols/cell-culture/transfection-protocol/rnaimax-reverse-transfections-lipofectamine.html), which is generally used to transfect much smaller nucleic acid molecules than what I want to do for this experiment.
Does anyone here have any experience with this topic? Do you have any protocols, or do you think I could modify the RNAiMax protocol?
I would like to cultivate Human Dermal Fibroblasts: HDF, neonatal (106-5n, Sigma Aldrich).
This type of cells are cultivated in the Fibroblast Growth Medium (116-500) according to the protocol. In addition to the classic Calf serum and antibiotic, is it necessary to use growth factor and insulin, as with other neonatal cells?
Or is there any alternative for it?
In our lab we work with human lung biopsies in which we digest and plate on fibroblasts to produce beautiful clones.
After the clone shave grown for 7-14 days we trypsinize and resuspend in StemCell Cryostor.
We have never had a problem when thawing and re-plating, but recently we haven't been able to regrow the cells after thawing to the full extent as the could be.
Taking any and all suggestions if you have run into this problem or if you have a protocol for freezing down and thawing lung epithelial cells that works wonderful for re-plating.
I am working with primary human fibroblasts from patients with severe metabolic disease. I am interested in using RNA/mRNA re-programming of these fibroblasts in order to obtain iPSCs for downstream functional characterisation.
In recent years, numerous kits/protocols have cropped up purporting to tackle this issue. I was wondering if you could share your experience of this approach and which method you found most effective?
I am trying to culture human neonatal fibroblast with a keratinocytes together to make an organotypic skin model.
Currently, I am working according to the established protocol: https://pubmed.ncbi.nlm.nih.gov/17401352/
The difference is that I had already used a created collagen solution: https://www.sigmaaldrich.com/catalog/product/sigma/c3867
But the solution does not transform into a 3D gel, even if I add 1M NaOH in it, or by adjusting the pH of the solution to a neutral range.
Is there any other problem, e.g. in collagen, temperature, ...?
Thank you for your answer!
I am isolating primary hepatocytes from mice. I am using the two-step collagenase perfusion method and after seeding the cells in the 12-well plate, I am seeing two types of cell morphologies, one with hepatocyte morphology and another with fibroblast-like morphology. Since according to my knowledge it's not possible to passage primary hepatocytes, it is difficult to get a pure culture after passaging. The alternative to this problem is that I sort the hepatocytes before seeding. But I couldn't find any marker specific for mice primary hepatocytes. We can't use the regular adherent markers such as EPCAM and all because it is present in fibroblasts also. So any advice or suggestion will be highly appreciated. Thank you in advance.
For almost a year now I've been trying to isolate osteoblasts with a pre-stablished and validated protocol. Basically I dissect calvaria from mice pups (4 days old) , then I perform 2 incubations with EDTA which I throw away and then I keep the following 4 digestions with collagenase B (0.2%). At first following this protocol I obtained a lot of cells but they were not osteoblasts, it seems I had fibroblast contamination, so we updated the protocol and I added collagenase to the EDTA step (so my first incubations were EDTA-collagenase) which normally helps for the contaminations. Only now I have a terrible yield for this protocol. It seems that I do obtain osteoblasts but too little, so little that I can't manage to expand them enough to perform tests later on (they are no longer in a good physiological state). With this protocol we should be able to obtain more than 200.000 osteoblasts, while I obtain in average 30.000.
I have technical problems but we can't identify them. There's a couple of post-docs in the lab (I am a PhD student) who manage to isolate osteoblasts, but they have supervised me and validated my steps and we can't seem to find the origin of the problem. Any ideas of technical steps I should pay attention to, or if anyone has had problems with osteoblast isolation in the past, did you manage to pinpoint the problem? Perhaps it could be my case or give me ideas on what to check. Thank you.
I am currently analysing some RNA-Seq data from human primary fibroblasts. I noticed in the following paper (https://www.nature.com/articles/ncomms15824) that the expression of hox genes was used as a proxy for biopsy site.
Would anyone have any potential scripts or other resources they could point me to? I'm just not sure how to code it/which hox genes to include.
Many thanks for your time,
I isolated exosomes form human serum using exosome isolation kit. I lysed the exosome pellet in 1X RIPA buffer with protease inhibitor. I also cultured the fibroblast cells and used the 1XRIPA Buffer to lyse the cell. I ran cell lysate and exosome in 12% polyacrylamide gel and transferred it to PVDF membrane.
I was able to detect CD 63 in exosomes and Calnexin in the cells. Cd 63 was not detected in the cells and Calnexin was not detected in the exosomes sample.
Now I wanted to detect loading control Beta actin and GAPDH in both of the sample, but I am only able to detect beta actin and GAPDH in the cell lysate and not in the exosome sample.
Could anybody answer why I am not able to detect these loading controls in my exosomes.
I tries adding 1X TritonX 100 to the sample, but it did not work?
While performing proteomic analysis of MEFs (four passages, P4), we have detected certain macrophage-specific integrins. Does anyone know if is common to have macrophages growing in primary MEFs cultures (passages 4-5), or if MEFS can express this type of integrins?
We had performed the method described in "Identification of HSP90 inhibitors as a novel class of senolytics" using human endothelial cells and human fibroblasts. Briefly, cells were incubated with 10 uM of C12FDG (2h), then prior to analysis, DNA were stained using Hoechst dye 2 ug/mL and we observed green fluorescence stainning in both senescent and non-senescent cells. We also tried to change the C12FDG incubation times and concentrations and we obtained the same results. We are using another kit to detect SA-BGal (senescence detection Kit ab65351) as a control method and the results do not match. We have the attention of always preparing a fresh solution of C12FDG when starting a new experiment.
We want to use this method for immuno fluorescent method to quantify senescence using INCell Analyser.
Do you have any suggestion that can help us?
I seeded the cells following the suggested density (0.8*10^4 cells/cm2). In 24h, few of them are adherent (low viability), and the ones that adhered are still round-shaped. Do you have any idea if it's normal that they need some time to spread and acquire their normal shape? I'm worried they went through some kind of stress during storage/thawing... Thank you very much for your time and help
I'm trying to test the effect of dust on lung cells
I was thinking of getting 2 conditions as my negative control, one without anything and one with something that mimic dust in physicals properties without the actual dust
the closest I can think of is titanium dioxide
What do you think? or should I just stick with one negative control i.e. no dust
I isolated the neonatal fibroblast cells from human forskin and I want to immortalize these cells by using plasmid and SV40 T antigen .
I do not have any experience in this regard if you have any , I really appreciate if you could share the tips with me.
I transfected my cells (primary human dermal fibroblasts) with a plasmid containing the gene of interest and a C-terminal turboGFP tag.
I could see the turboGFP fluorescence using a fluorescence microscope.
So I fixed these cells in ice-cold methanol and stored them at 4 °C, covered with an aluminium foil.
However, when I performed an immunofluorescence, the green fluorescence of the turboGFP almost completely disappeared.
Does the GFP fluorescence disappear in fixed cells?
How long does it last?
Here's some background for my question. I have a suspicion based on literature reviews and recent RNAseq data that defects in vitamin D metabolism may be contributing to the observed phenotype between my diseased and control cells. I'm working with a system of cultured human fibroblasts and would like to test out my hypothesis by using qrRT-PCR to quantify the transcript levels of key genes in the vitamin D metabolism pathway coinciding with assaying levels of D3 and 25(OH)D (i.e. calcitriol).
I'm doing my google research right now but would appreciate insight from anyone with experience assaying vitamin D levels in cultured cells. What assays/methodologies would you recommend? Are there any beginner pitfalls I should Be particularly cognizant of
I have HDF cells and I grew them with DMEM but when I look at them I can tell that they are growing slowly. I read that there is a complete fibroblast growth medium (ready to use) but it's very expensive. any suggestions on how can I get a better medium? I have DMEM, RPMI, L-15 and EMEM.
thank you very much for your help
I am sharing this here as it does not appear to have been shared previously. I hope it eases the anguish of anyone else who has made the same mistake in their lab work wondering what is the fate of their dry ice frozen stored fibroblasts!
Last week I accidentally froze fibroblasts (in 90% FBS: 10% DMSO freezing media) using dry ice instead of the Mr Frosty device. Within an hour of being frozen they were stored in vapour phase for a week before being thawed in a 37c water bath and plated out.