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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?
Thanks. Carlos.
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Thanks for your reply.
Do you know how long it takes for cells get stressed? I have read about 10-30 minutes of exposure time with H2O2 at concentrations of 20-100 uM.
Do you know if after 10-30 minutes of exposure time, cells are already stressed to perform the assay?
Thanks.
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Hello Guys,
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.
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Thank you Venus Dilshad
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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.
Images attached.
Appreciate your help
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Hi Maria,
So, the trouble with primary cells is that you never really know the status of the donor. I agree with Gayatri, in that your fibroblasts don't look healthy. There are couple of things that could be going on here:
  1. Early stage mycoplasma contamination: sometimes, mycoplasma will give a false negative if it's early on in the infection. In my experience, PCR based assays are more accurate than colorimetric assays. If the media looks "sandy" and the cells grow much slower than normal, that's usually a sign of mycoplasma.
  2. Bacterial contamination: this will slow down your culture and rob your primary cells of nutrients. You can streak some of the conditioned media out onto and LB plate and grow it at 37 C overnight and see if any bacteria grow out.
  3. Viral contamination of the host: these cells might be harboring an unknown virus that the host had. This isn't likely, but it's a possibility. I don't know of anything you can do if this is the case.
As for what you should do next: I suggest that if you have an earlier passage frozen or can get another source of primary cells, that you start over. I realize that with primary cells, your source might be from a surgical sample, so this might not be an option.
So, if these are extremely precious cells, you can grow your cells with a higher/secondary antibiotic in your culture media for a while to try to "cure" your cells of mycoplasma and/or bacterial infection. You can get a good anti-myco reagent here: www.invivogen.com/plasmocure
This process takes around 1-2 months, unfortunately.
Also, makes sure to rule out any obvious culprits: make fresh media, make sure you have all of your correct media additives, decontaminate your incubator, etc.
Anyway, I hope this helps. Good luck in your research!
All the best,
Bryan
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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.
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I grow primary lung epithelial cells and use TryplE to lift the cells.
I don't really have a problem with the "long" incubation time (I use 5 minutes), however sometimes when my cells are particularly well adhered I need three incubations (this doesn't seem to affect viability particularly).
In saying that, I've not used accutase much, so I can' really comment on the comparison of the two.
Best of luck!
Sam
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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?
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Subhash C. Juneja Thanks for your reply. I found that the membrane of transwell seems to be unidirectional because once the medium leaked, the upper chambers were nearly dry. This affected a lot to the ADSCs in transwells. Maybe I should change the transwell to other brand's.
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Dear all,
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.
Or,
is there any possibility that these cells would make a differentiated skin model without a solid collagen layer below them?
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I think that the data sheet for that solution says that is not suitable to 3D cultures as it has been sterilised using UV light, that eventually can interfere with the cross linking that is necessary for 3D cultures
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Can we differentiate ipsc grown on MEF ( mouse embryonic fibroblast) to cardiomyocytes on MEF itself?
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for iPSCs grown on MEF most of the labs use EB formation / hanging drop method- https://www.ahajournals.org/doi/10.1161/circulationaha.109.868885#d3e287
doi:10.1093/eurheartj/ehs349
Also pls have a look here for more detailed comparisons of differentiation protocols-
MEFs in general do not give very reproducible results. If you really want to use MEFs then you could also think of trying MEF conditioned medium- https://www.nature.com/articles/s41598-018-24074-y#Sec10
good luck
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Hi everyone,
I isolate tenocytes from mice tendons. I extract the mice tendons, place them in collagenase overnight and then seed them on T75 flasks. I have been using the same medium as the people before me (DMEM42430-025 +10%FBS,+1%P/s+1%NEAA).
The cells were already looking strange in the previous isolation so I seeded them on collagen coated flasks this time. They still seem to differentiate into some kind of weird cells.
I was wondering if anyone has any experience working with primary cells and have observed this before?
Thanks!
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Thank you Ellen A G Chernoff and Subhash C. Juneja for your replies! I was worried because I thought they should be more fibroblast like but now i understand that when i digest the tendons directly in collagenase, i get a mixed population of stem cell-like (polygonal cells) and fibroblast-like (which would be "tenocytes").
I will check out the other method: fibroblasts directly coming out of the tendons to get only fibroblasts.
Cheers!
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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?
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From the image, I can observe very few cells. What is the seeding density you are using when you subculture rat dermal fibroblast?
The intercellular signaling mechanism is largely determined by the cell population as well as the surrounding environment. The cell seeding density can influence viability, proliferation and differentiation of cells.
Seeding a lower cell density will result in lack of cell-to-cell interaction and communication. As a result, the cells will show slow growth and eventually die.
On the other hand, seeding a high cell density than the optimum will result in cellular stress as there will be overcrowding and lack of space for cells to interact with each other and grow. Moreover, nutrients in the culture medium will be insufficient and there will be accumulation of metabolic waste causing cells to face stressful conditions.
So, use an optimal cell seeding density when you subculture the cells. Generally, for rat dermal fibroblasts you should seed 0.5 to 0.8 million cells in a T-25 flask.
Hope this will help!
Good Luck!
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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.
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These kinds of cells are common in L929 cell lines. You can refer to the links below from ATCC and Riken :
These could either be terminally differentiated cells or senescent cells. You can get rid of them when you passage the cells, they might most probably remain firmly attached to the plate.
You don't have to worry as long as these are low in numbers.
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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?
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In my case, I cultured gingival fibroblasts till 70% connfluent then stimulate with Pg LPS 1microgram / mL (final concentration) for 24 h. mRNA induction and ELISA results were good. I used ALpha MEM with 10% FBS and 1% p/s
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Hi all,
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.
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Hi!
We prepare our loading dye ourselves and apparently, the wrong amount of B-ME was added. So after preparing new loading dye, the problem was solved. We noticed the LD was the problem after we borrowed commercial LD from another lab. I hope this works for you too!
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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?
Thank you
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If it is normal cells (not infected with any virus or bacteria, in that case we can't change the time and concentration of PFA), you can reduce the PFA concentration and fixing time as well. This would greatly preserve the GFP fluorescence.
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Hello!
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!
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Hi!
Pooja Rani Mina : DNaseI is added after the trypsin digestion, not the collagenase.
Shilpa Bisht : In the end, what works the best (at least for me) wasthe lamina propria dissotiation kit from Miltenyi.
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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
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The trick with removing cells whilst retaining an intact ECM is to do every step extremely carefully. Always pipette in and out at the same position against the side of the culture dish and one drop at a time. It takes forever but it worked for me. Also, how long are you allowing the fibroblasts to produce the ECM? I remember seeing in the literature people generally waited 10-12 days but I got much better results when I waited for about 21 days (my aim was also to culture cancer cells on the ECM).
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Hello,
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.
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The primary translation product for bFGF is composed of 155 amino acids without a traditional signal peptide. The mature form of secreted human bFGF is 146 amino acids long (nine amino acid residues at the N-terminus are likely proteolytically cleaved from the 155-aa precursor). The biological activities of 155 aa form and 146 aa form are similar when tested under the same conditions, indicating that the N terminal region of bFGF is not involved either in biological activity or in binding to FGF cell surface receptors. Although there is no difference in biological function for these two forms, it is recommended that the 155 aa form be used for ES cells and the 146 aa form for neural cell studies.
For more information on bFGF you may refer to the references given below.
Best.
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Dear All,
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?
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Have you tried coating the plate with some ECM component so epithelial cell can re-attach? Some papers says laminin enhances epithelial cell attachment.
So you could try. Good luck.
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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!
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Okay. Good observation and well done Muhammad Elezzabi
Good Luck!
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Hi there,
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 :-)
Kind regards
Miriam
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The mean number of cells dissociated per cm^2 biopsy after trypsin: EDTA digestion of a dermis-containing biopsy is 4.0 x10^6 cells/cm^2
Between 0.5% and 4% of the cells dissociated from a dermis-containing biopsy are human fibroblasts. For more information you may refer to the article attached below.
Hope this reference is helpful!
Best.
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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.
Thank you.
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Hi Lëonid,
Once happened to me and searching in literature I found some articles relating cAMP with this morphological change (PMIDs 2993305, 232579, 17557277, 20840080).
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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
31.2.22
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.
25.1.22
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.
17.1.22
pMEF 1 embryo wt. 0.70 TC 5% 37’C, all ok, frozen down. cMEM 14.1.22. all seemed ok
10.1.22
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.
23.11.22
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.
03.11.21
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.
06.10.21
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.
27.09.21
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.
20.09.21
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
08.09.21
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)
31.8.21
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)
20.07.21
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.
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Looks like contamination to me. Have you checked all solutions that touch the cells for contamination? Even the trypsin? Are you sure your frozen cell lines are clean?
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Hello everyone.
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?
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Try either
0.5mg collagenase A (Signa) /ml and 1.5 mg pancreatin/ml and incubated for 90 minutes at 37C in HBSS. While gentle shaking
Or
Make very small pieces of the tissue (2 mm approx), leave in a dish with culture medium with FBS and Pen/Step (do not dip tissue in the medium, change medium carefully from the side (remove old medium and add fresh medium) every 3 days. The fibroblasts will proliferate in 10 to 15 days, you should get monolayers in less than 15 days. It is slow but non-enzymatic method, I have done in many tissues, it worked
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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?
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Are your cells under selective antibiotic pressure?
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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?
Thank you
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Hi Mina,
appologies, this answer might come a bit late, but our tech support team also has some thoughts on your questions:
The ibidi Polymer Coverslip and the ibidi Glass Coverslip provide ideal optical conditions for fluorescence microscopy. Therefore, confocal microscopy is possible without restrictions when using any of the ibidi labware that contains the ibidi Polymer Coverslip or the ibidi Glass Coverslip Bottom. A suggestion would be to pre-coat before adding the gel - we do so for the µ-Slide I Luer 3D. This is done to enhance the attachment of the gel to the surface of the slide. A suggestion could be to use a collagen coating, that might bind nicely with the collagen fibers in the gel. An example of this can be found in the instructions of our µ-Slide I Luer 3D instruction page 2: https://ibidi.com/img/cms/products/labware/channel_slides/S_871XX_Slide_ILuer_3D/IN_8717X_Luer_I_3D.pdf
An alternative would be to try using one of our µ-Slides with Polymer Bottom instead of class. The coating and the gel would probably work better on the polymer coverslip, and as mentioned before - the optics for confocal microscopy are the same as on glass.
You could also switch to a slide format with smaller wells (e.g. the µ-Slide Angiogenesis or µ-Slide I Luer 3D). The wells in these formats could create a stable environment for the gel and have volume-dependent savings, and maybe even more wells pro slide/dish.
Regarding the cell number it is difficult to give a generalized recommendation. The cell concentration is very much dependent on the cells, the total volume, the format used for seeding and the assay. We always recommend setting up specific assay criteria based on the literature and determine a seeding density based on that. We recommend ordering a free sample of the µ-Slide Angiogenesis and test 5 different concentrations in triplicates. Please remember that the cell suspension should have quite a high cell concentration since it is diluted in the gel e.g. in our Collagen I protocol it is diluted by a factor of six.
I hope this helps.
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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?
Many Thanks,
Fouzia
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In monolayer cell studies, CM is really nutrient depleted medium. This nutrient depletion causes the monolayer cells to spread out more. I.e., become flatter, which causes their nuclei to flatten and changes the chromosomes to change their distribution from 3D to 2D and hence, the number of their nearest neighbors. This halves the number of neighbors for chromosome exchanges and other interactions. The same effect can be produced by diluting fresh media with saline. See the my papers with N.M.S. Reddy, with Peter Mayer, with Joseph Ostashevsky, and with Maria Kapiszewska, all in the 1990s and early 2000s.
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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.
-Tim S
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To clear your curiosity, let me tell you under culture conditions, healthy cells will show all physiology including adhesion to the substratum (for adherence cells/cell lines), elongation, spreading and division etc.
If the cells are starved (if cultured in serum free medium) or exposed to sub-optimal conditions that can be easily observed by reduced attachment, elongation, spreading and focal adhesion points.
On order to minimize the exposure of insult/stress, cells under in vitro conditions show various morphological changes and try to reduce the surface area. Hence, the cell adhesion, elongation and spreading properties of the healthy cells are adversely affected.
If the level of insult/stress is more then you can also see the cytoplasmic granulation, cell shrinkage, membrane blabbing, and even cytoplasmic fragmentation in some of the cultured cells. Actually these the morphological features of apoptosis in stressed cells.
You can see some of the cells in the image are showing good attachment, increased cytoplasmic area and spreading with number of focal points, indications of healthy cells.
Best
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Hello.
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?
thank you.
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Hi
I think that the problem lies mainly in the confluency and the number of cells seeded per Well.
Also, you must pay attention that the fibroblast expands as it grows in shape and their size will be large, but the number is less.
The conclusion is that compared to other cancerous lines, fibroblasts expand more in size than their number that make them die on the border.
Best regards
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I want to do a direct co-culture of cancer cells and fibroblasts. After doing co-culture, I want to get back my cancer cells and fibroblast for further downstream analysis. So, how to separate them after co-culture?
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Dear @salam
You may separate the populations by sorting (FACS or MACS) for further analysis.
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Dear all,
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,
Marissa
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I'm sorry I don't have direct experience with that. Good luck!
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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.
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The colonies could be a mixture of pluripotent stem cells and differentiated cells. Usually, when iPSC/ESC colonies are maintained longer in a single culture dish more than 10 days they tend to differentiate. I would suggest, using TrypLE and dissociating the colonies to single cells and seeding it again. From there you can pick colonies with proper iPSC colony morphology and expand them.
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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!
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Vector Builder is great if you want to customize further, or this one:
Try multiple viral titers for 6-8h.
All the best!
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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?
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Yes the center had differentiated.. Try passaging the out ones..
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Hello,
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?
Thanks
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Thank you everyone for your recommendations and your time.
I realized doing subculture when they are not fully confluent definitely helps a lot!
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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.
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Hi @Michael Phang
I would like to know if your problem got resolved or not.
Because, I am getting the same problem since last three MTT experiments. Even after observing cell death under microscope, I am still getting high OD value after performing MTT assay.
Please let me know how did you resolve this issue!
Thank you in advance!
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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.
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Hello everyone!
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.
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The above links include a single-cell RNA seq that may be helpful. Good luck.
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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,
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@Diana Baxter
Thanks.
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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?)
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Hello Yuhao Wan
2500-5000/cm^2 is suggested if you are not planning to make any sort of manipulations right after so that you avoid as much as possible trypsinizing and splitting your cells. Meaning that if you are not planning to do any transfections or drug treatments on the day of plating (or consecutive day of platting).
Based on my experience with fibroblasts (MRC5, CAFs, and Human primary prostate fibroblasts) I found that 100,000 cell/9.6cm2 (6- well plate) is the best if you are planning to do an experiment on the day of platting or on the consecutive day. And then for sure, you scale up and down based on your culture plate and the surface area you are using exactly.
I hope this was helpful for you
Good Luck
Lina
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I just got a new stock of NR-6 cell line (Mouse embryo fibroblast cells that don't express EGFR) and I need more information about the culturing and specifications of it.
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The above described the culture media. The best is to ask the company or lab you got the cells. They are the ones who culture and make the stock. Alternatively, you can search using PubMed or another. Good luck.
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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 ?
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Two questions for you:
  1. How did you differentiate your cells?
  2. What cytokines are you interested in seeing?
E. coli LPS (I use O111:B4) at a concentration of 1-10 ng/mL will induce several cytokines to release from macrophages within a few hours. A great example is TNF. However, other cytokines will be expressed but not released, such as IL-1b. It will remain intracellular upon LPS stimulation until induced to release by an inflammasome activator.
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I am looking for RNA sequencing data of dermal fibroblasts and CD34+ HSC performed using the same sequencing kit. I am unable to find such data. Can anyone suggest where can I download it?
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Dear all,
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?
Best regard
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Dear Dr
I think that it is better to use collagenase enzyme. or If you don't have collagenase enzyme you can use a magnet and trypsin enzyme for 5 minute then culture the tissue particles in plates. I hope this methods help you.
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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.......
Thank you!
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Thank you Mario Romani i Will read it.
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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.
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Hi Yeaji,
I agree with Ankita that a cell-free control should be run to determine if the scaffolds are giving a positive signal with the assay. We have not had an issue with high baseline cytokine release in our internal testing using our SeedEZ scaffolds, although our work with fibroblast has been limited. I'd be happy to help you further if you provide some specifics about your experiment. Please reach out via our website and I will answer you personally.
Best wishes,
James
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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.
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I think, it is important to know how are you preparing the fibroblast.
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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?
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Michael Davis has answerd this by saying reverse transfection is the answer and I do agree with him and the paper shared by the expert which also shows about the details of Transfection Reagent for Fibroblast Cells.
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What concentration of camptothecin is effective to kill mouse fibroblast in 24h?
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Lydia Amari , you should always run a pilot experiment treating your cells with different concentrations of camptothecin. You can assess the viability by either trypan blue exclusion assay or MTT/MTS (Cell Titer) assays. You can use the doses in the literature as a baseline, but that can drastically change depending on your regents (e.g. different brands/purity concentrations, medium supplements interference, etc) and culture conditions.
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Dear all,
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?
Thank you!
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Thank you for your advices!
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Hi all-
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.
Thanks
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Dear Kiana,
It seems you are using a primary culture, isolated by yourself from the biopsies. The properties of primary cultures are much different from established cell lines. Primary cultures usually exhaust after several passages. They also may not withstand the harsh condition of freezing for several times. However, it is a undeniable fact that cells are hard to regrow after long-time freezings and you should be patient with these cells. some times they need 2-3 weeks to regrow and become refresh. My suggestion to you is being patient with the cells, even if you only a very low cell number in your culture vessel. I also suggest increasing the concentration of PBS in your medium to 15% and using glutamine supplement. This helps your cells to regrow faster.
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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?
Many thanks,
Rob.
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Hi Jing Liu . That sounds very promising. I will definitely check it out. Thanks for your answer!
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Dear all,
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!
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You are most welcome Kristýna Valášková
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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.
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Hello Vishal Sah
You can use cytokeratin-18 found on the extracellular surface of hepatocytes.
Please refer to the article attached.
Best.
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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.
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How long do you incubate in collagenase or EDTA?
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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,
Rob.
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I think your answer is in the supplementary materials of this paper that you have cited https://static-content.springer.com/esm/art%3A10.1038%2Fncomms15824/MediaObjects/41467_2017_BFncomms15824_MOESM399_ESM.pdf on pages 25-26.
The authors have used the expression of hox genes from all the samples to cluster that data matrix and then used those clusters to create a new factor variable with x levels and used it as an adjustment factor in the regression model.
If you are asking how to create this cluster variable, then one way would be to use the 2 functions in R, you can google for a detailed usage
hclust and then cutree
You can also see a quick example from one of our project code
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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?
Thanks
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Hello Kamala Lamsal You will not detect Actin in exosomes unless it is being packaged in them. Exosome cargo are veriable so using cytoskeleton markers like tubulin and actin is not adequate. As for GAPDH the same principle applies some will have it if it is packaged but it can also attach to outer surface of exosomes which means it may be lost during isolation/lysis procedures (doi.org/10.1038/s41467-021-27056-3). It is best to choose a loading control that is part of the membrane make-up of both cells and exosomes such as Flotillin -1 and Alix or AIP1. Depending on type of cells you are working on CD9 may be an option too. Good luck.
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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?
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Hi, you shouldn't observe macrophages in MEF. As Ellen stated, there is overlapping of integrins between cell types. Re-check with specific markers of macrophages and you may share images with us if you want. On another hand, you may ask the other researchers using the same batch of MEF.
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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?
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C12FDG is a substrate for Bgal enzyme. Any normal cell which is treated with the C12FDG substrate will show green fluorescence because BGal in the lysosome is highly active at pH4.0. A senescent cell has more of this enzyme so you first increase the pH inside of the cell to 6.0 by adding Bafilomycin A and then add C12FDG. Now whatever green cells you see are apparently senescent cells.
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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
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Hello, I am currently having the same problem. It's been 3 days since I cultur@ed the cell, but it still didn't stick to the flask. How is the condition of your cells? Is there a solution you can suggest me?
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Hello,
I am looking for a specific antibody to stain Fibroblasts in a live staining and to distinguish them from HUVEC cells. Does anyone know of such an antibody I could use?
Thanks!!
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Tahir Detinis Did you manage to stain fibroblast in live staining? I am having the similar problem.
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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
Thank you
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Maybe you can try using Silica dust. It will also serve the purpose of dust. Being inert, it will not affect your medium. Also, you may try to collect some dust naturally and subject it to autoclaving after thorough packing into a durable container such that moisture does not enter it. This way you can sterilize it and use it for further experimentation. Hope this helps you.
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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.
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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?
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GFP fluorescence does not work after methanol fixation, however it should work fine after formaldehyde fixation.
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Hello,
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
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The conversion of vitamin D to 25(OH)D (i.e., 25-hydroxyvitamin D) occurs in the liver, so I am not very sure about the fibroblasts. A general description is here:
There are some studies investigating the effects of 25-hydroxyvitamin D on fibroblasts:
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Which medium should I use for this experiment? it should contain no phenol red.
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Hello,
Human fibroblast cells are cultured in DMEM +10%FBS.
For your experiment you can use DMEM with low glucose and sodium pyruvate, but without phenol red and L-glutamine.
This medium is available at Thermo Fisher Scientific (Gibco)
Cat# 11054001.
Best.
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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
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Hello Nili,
We culture our HDF cell line in DMEM (Sigma, D5796) supplemented with 10% v/v heat-inactivated fetal bovine serum and 1% v/v sodium pyruvate. They grow pretty well in that medium.
I advise you to use the media that is suggested by the supplier. Otherwise, you can adapt the cells to a new medium by gradually replacing the old one with it. Just keep in mind that changing the culture medium may have some implications on several levels (RNA/Protein expression, metabolism, etc.).
There are several possible reasons why cells grow slowly, such as high passage numbers, microbial contamination, different FBS batch, cells are too diluted when subcultured, etc.
You will find below some useful links regarding poor cell growth:
I also found researchgate questions that may be helpful:
Sincerely,
Y.S.
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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.