Primary Cell Culture - Science method

Hi,

Then it is absolutely normal for your cells to adhere to the culture flask. If you're still maintaing them then I would suggest you to split them in 1:3 ratio to be on the safer side.

In this case, the color change is due to an increase in pH and not contamination.

Doesn't seem like a contamination, but more of a poly(propylene)fiber filament, a result from plastic injection of pipette tip during production. We usually ignore it, through centrifugation it will disappear, eventually. Else keep an eye on the tip, if one showed with filament seems going to detach, please discard it and choose a new one.

I aslo think that checking the purchaser information for cells/media would help. And, addition of L-glutamine to medium already containing l-glutamine is mostly not necessary.

Hi Adenine Koo,

You can use the same CO2 incubator and biosafety cabinet for working in both primary and immortalized cell lines. However you should use special precautions while working with both the cells. Do not use both the cells together while working in biosafety cabinet and do not open the caps of the flasks at the same time. Sanitize all the plasticwares packs and surface with ethanol.

Best of luck.....!!

Thanks

There may be some red cells in there. You can add a 30 second water lysis step. Lyse cells in water and then add an equal volume of 2x pbs. There looks to be some dead cells and debris so you may also want to wash the plate with PBS after the plating step. If you are still having problems you may want to look into a monocyte isolation kit like Miltenyi.

Hi,

Let me know if your experiment worked and if possible please send me the detailed protocol. I am trying to set up a primary culture of intestinal epithelial cells from mice.

Kavitha Jade Brunner it sounds to me like you're doing the right things regarding the water in the water baths. As for cleaning surfaces, using the bleach wouldn't cause contamination, it can just wear down on the surfaces over time.

Disinfecting the scope and surrounding areas as best you can will likely help quite a bit! I'd love to hear if it helps as I am quite curious as to your contamination source as well, now.

I'm not sure why you don't filter media, but perhaps there is something about these cells/this media that I'm unfamiliar with, which is certainly a possibility! Since you're using antibiotics, it can be good to regularly test for mycoplasma as well if you don't already. Though you may not have visible contamination in all your flasks, contamination can sometimes be masked by antibiotics. It seems to be a point of contention among scientists (much like "a-POP-tosis" versus "a-PUH-tosis" haha) but I personally prefer to culture cells without antibiotics. If your aseptic technique is good (sounds like yours is - your source of contamination is likely from the dissection conditions), there's really no need for antibiotics in my opinion! Your PI may vehemently disagree, but that's just my two-cents. Good luck and feel free to keep me updated as to whether the scope disinfection helps!

The n = 1 with 6 technical replicates because you processed all of the embryos into a single tube before plating. If you had processed each embryo and plated the resulting cells separately your 'n' would be 15.

Hi Vincent,

You can test the cells for senescence markers using a number of kits.

To easily and rapidly lyse red blood cells adhered to macrophages,

I use an ACK solution (made of ammonium chloride is a buffered solution, prapared in house or commercially available, too), for 5 min. at room temperature.

Macrophages can resist such treatment with ACK but not the red blood cells.

Definitely a fair assessment, Ive made other samples that have similar properties as this, but none of the other samples Ive generated have responded with as much vigor as this sample.

Definitely will invest in generating another panel of T-cells but from what I can tell so far, this sample had a particularly rare phenotype that i may or may not see again in a relatively limited panel size. every once in a while I remember I have this last vial and wonder if I can do anything with it, in the end its always for the birds...

Hello Fouzia Zayou

For primary cells isolated directly from tissues, or their early passages are at a particularly high risk of contamination as the dissection process may promote contamination from the natural commensal flora in certain tissues and any subclinical infection. For cells isolated from primary tissue, it is necessary to use antibiotics in the primary culture. Antibiotics should be removed as soon as possible, and the culture should be tested for mycoplasma after at least two passages in the absence of antibiotics.

Please refer to the link below for preventing contamination of primary cell cultures.

Hope this helps.

Best.

Dealing with primary cell culture can be tedious sometimes and of course, they are not easy to deal with.

We specialize in producing high-quality primary cells. You can connect with us at info@kosheeka.com to know about primary cell culture or you can visit our website https://kosheeka.com/ for further details

depend on the lower limit of detection (LOD) of the PCR, the viral load, and the stage ( or clinical feature) of the patient

Diana is right, you have to isolate islets immediately. In our lab we use the protocol that we just updated in this publication to isolate islets.

For the WB: I collect about 100 islets that are similar in size and wash with PBS, spin and collect PBS. Add 30uL of SDS sample buffer and boil for 5min (basically you are lysing your islets in the running buffer to avoid dilution). Load 12-15uL on a western blot lane (I use Bio_Rad mini gels.) If you use islets of similar size, I don't have problems with unequal loading.

@benedetta colmegna

Hi, I have the same question. Could you let me know the protocol you finally ended up using?

if your media has changed color but not turbid (indication of bacterial growth), your cells have exhausted the media. Phenol ref in the medium is added to see the physical appearance of culture so as to change/replenish the medium.

Hello Paulius Valiukevicius

You may also refer to the article below.

Best,

Dear Benjamin Tschumi

If you have drawn the sample in the sodium-heparin tube then you need not to add further Na. Sodium in the tube is enough to stop coagulation.

Even if the coagulation will occur it will occur in 30-40 minutes. Check with control sample and then think of adding more Na to the tube.

Hope this is helpful.

Good luck.

Hello Natalie Daw

FBS may vary in quality in terms of growth factors, amino acids and other components from batch to batch. Initially the cells may not show the effect of the new batch of FBS because they already have stored the necessary growth requirements for their growth from the previous batch of FBS. Once these stores are depleted the detrimental effect of the new batch of FBS if any becomes visible because the cells will be utilizing the new batch of FBS.

So I suggest you culture your cells in the new FBS batch alongside the present usual batch of FBS in which the cells are growing fine. Compare various parameters such as cell viability, morphology, etc. just to see that the new batch of FBS does not have any detrimental effect on the cells. If everything works fine you can grow your cells in the new batch of FBS.

I hope this helps.

Best.

Hello,

As Joseph mentioned, you have to be careful for how long you´re warming the cells, thawing freezed cells has to be a fast process, since DMSO is toxic for cells and can be the reason why you have low percent of live cells. Remember to check for any thawing specifications regarding your cell lines with your supplier.

Basically, both Freshney´s Culture of Animal Cells textbook and Thermo Fisher Scientific´s webpage recommend the following general thawing protocol:

1) Remove the cryovial from liquid nitrogen or -80°C freezer and immediately place it in a 37°C water bath. 2) Swirl the cells until you see a small bit of ice in the cryovial (this shall take about one minute). 3) Transfer the cryovial to a laminar flow-hood, open it, and transfer the thawed cells to a centrifuge tube. 4) Add pre-warmed medium appropriate for your cell line in a dropwise matter. 5) Centrifuge the cell suspension at approximately 200 g for 5-10 minutes (this vary with your cell line, I recommend you to check). 6) Decant the supernatant, resuspend the cells with medium, and transfer to a culture flask.

Note: some cells can be thawed without the centrifugation step, you can dilute the cryoprotectant with fresh pre-warmed medium, but you must change the medium the day after thawing.

Suggestions: make sure to work fast but aware of the steps of the process; while thawing the cells in the water bath make sure not to submerge the cryovial since this can increase the probability of contamination; try not to thaw cells that were freezed a long time ago, since viability can decrease; remember to always work aseptically; double check the label of the cryovial to make sure of the identity of the cells; plate thawed cells at high density to increase recovery.

In case you want to read more about thawing, Freshney´s textobook is very useful: Freshney, R. I. (2006). Basic principles of cell culture. Culture of cells for tissue engineering, 3-22.; you can also visit Thermo Fisher Scientific website, which has a video and a guideline regarding the thawing process: https://www.thermofisher.com/cr/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/thawing-cells.html

Sarah Hélène Merkling - vivoPHIX sounds really interesting, have you used it yourself to process tissues + dissociate for scRNAseq? Would you mind sharing your experience with it? Thank you!

A gel substance like substance as media. It must contain nutrients for the B cells. Should you not stimulate the cells from bone marrow? Maybe from small age. The stimulation could be more of human.

Its really a very simple answer!

1. Serum which you use as a survival supplement is an alarm signal; it indicates tissue injury (serum is the liquid component of clotted blood).

2. Serum proteins adhere to the culture vessel surface, providing cell attachment ligands (recognised by cell surface integrins). Integrin engagement, in turn, facilitates cytoskeletal organisation (aka stress fibres), of which SMA is just one element. Given TC plastic is rigid, this provides mechanical resistance, that stabilises the actin cytoskeleton.

You see, its really simple, once you look carefully at what is in your system!

Dear researchers, thank you all for your contributions. Dear Mohana Krishna Gopisetty, the method you have suggested is time-efficient and problem-solving, I appreciated that. However, the cancer type we are working with is a little bit struggling. Our drug of interest is one of the members of the chemotherapy regimen which consists of other different drugs (we can say that it is given as a cocktail). Also, the relative survival rate of patients is low, diagnosis with specific drug chemoresistance takes time. (but we know that chemoresistance happens - under investigation)

One very basic tip depends on the % CO2 you are using. Without serum, DMEM has a bicarbonate concentration that relies on 10% CO2. If you want to remain at 5% CO2 go to a DMEM/F12, 50/50 medium, which has the appropriate bicarbonate for 5% CO2. Past that, I have not worked with microglia, but trying less time in Ara-C is something to try. Good luck.

Dear Agnieszka!

If you have done all these procedures and did not find contamination, according to the photographs, these may still be dead cells due to the type of apoptosis, when small apoptotic talci are formed. Based on your description, cells do not grow, do not attach, but die. Apotosis is a physiological, programmed type of death, and therefore your cells do not like any factors. Check media, serum, supplements to ensure all reagents are fresh. check the culture dishes (dishes, plates, vials), you may have used other manufacturer's dishes with different adhesive properties. Primary cell cultures like to grow on highly adhesive plastic.

One more thing: Your cells can just get senescence. In this case, their adhesion also decreases, the expression of surface markers changes, and increased death is observed. In this case, it is better to replace the culture, to defrost new cells.

Miriam Stölting Hello, we have been trying to isolate fibroblasts from the fresh human pancreas but haven't had a bit of luck. May I know the process and the medium you use in the process? Thanks.

Hello

I not exactly sure what aspect of D1 and D2 receptors you are interested in. For example, protein expression levels, mRNA expression, cellular location etc, or simply to mark particular types of cell. It sounds like you are interested in labelling D1 and/or D2 expressing cells, perhaps with a goal of quantifying how many there are, or some change in distribution or cell morphology.

There are plenty of companies selling anti D1 or D2 antibodies that may work for immunohistochemistry and I have seen publications looking at least D1 expression in rat cultures from the striatum (PMID: 11320256). My experience of performing this type of staining is to just try it as antibodies can be hit or miss. Unfortunately, they are expensive for just a pilot and you may try asking the company for a sample. Also, on this issue I find that staining in cultures can be particularly error-prone with regard to achieving a genuine signal as there is not as much background material to compare to. With this in mind, some kind of control is a very good idea, such as antibody blocking, or even better, testing the antibody in tissue from D1/D2 knockout mice.

Aside from immunohistochemistry, there are many other ways you can look in on D1 and D2 depending on your question, e.g. Western blot, RT-PCR, receptor binding etc. There are also mice with fluorescence-tagged D1 and D2 receptors (e.g. PMID: 28436559), though clearly introducing them into your study may be a much more involved and costly affair. That said they deliver an excellent degree of confidence in the legitimacy of the expression.

Measuring expression of TH is as you say common. I would expect it to be far easier than D1 and D2 by immunohistochemistry, but it is quite a different but related question. Again, I am not sure what your goal is, but measuring TH is commonly used for identifying dopamine neurones, but do remember that TH is also involved in the production of norepinephrine and epinephrine so depending on from where you are taking the cultures, you may need to consider this.

Good luck!

Niall

Have you sold this problem and what is the source of contamination? We have the same problem in our lab..

Ok you should probably have more than 84ng of RNA in total. I'm a little confused - you lysed the cells and then spun down the media? The media should be removed before addition of the lysis reagent. My protocol for RNA extraction from cultured neurons is roughly:

1) Remove and discard all media

2) Pipette required volume of lysis reagent into each well

3) Gently rock/tilt plate to ensure lysis reagent is in contact with all cells

4) Insert pipette tip into first well, vigorously stir the lysis reagent in the well and pipette up and down to make sure all cells are lysed and contents suspended

5) Transfer all liquid from well into a microcentrifuge tube

6) Repeat 4 and 5 for every well

7) Vortex all tubes thoroughly

8) Proceed with RNA extraction according to kit instructions.

I would not spin down the samples between collection and loading them onto the columns unless the kit instructions say to do that.

no I never worked with fixated tissue extrinsic fluorescence. sorry, i misanterstood you problem, cannot help further, thanks for your answer to my coment, Vera Lima

Thanks very much, we will try this and see what we will get

I seems like it's PC-3 !

Stimulation with CSA/ PhA and kept in CO2 for 3 -4 day with RPMI+15% FCS. after that at least 50-65% proliferation will found.

It's very difficult to handing also during this period contamination of culture is very high changes because of primary culture.

Dear Rahul Kumar by using DNA dye you will be able to separate G0/G1, S and M phase but if you add RNA dye as well, you will be able to separate G0 and G1. Attaching you an article showing protocol and gating for the same

Hello Marta,

The survival of P1-P2 DRG neurons will depend on the cell culture media you use and the presence of trophic support. Adding NGF to this cultures will help you to keep these neurons alive, but may not really fit your experimental protocol. More mature DRGs (e.g. P30) are less dependent on trophic factors for the survival.

Judging from the product description the exosomes are removed by ultracentrifugation of the serum without heat application. In this case, natural IGG's are still present in the serum and its heat inactivation may improve your culture growth. Even if you perform double heat inactivation of the serum it will not render it's useless slightly degrade it's quality at worst. You can also prepare a small amount of the media with heat-inactivated serum and without and see which is best for your cell's growth.

Hi, I'm also encountering such problem in my cell culture. So the whitish cells are bacteria or just debris? Thanks.

Hi all. I am planning you using beta-estradiol on agar plates for yeast. Any idea how stable will it be there? Thanks

Plesae type your question in google. You could find a lot ad answers then, you have to select easy one.

Dear Ori

I am trying to isolate B lymphocytes from bursa of fabricius for studying lymphocytes proliferation assay. After seeing your answers and tips that mention cells with low viability, I wonder whether it is feasible for lymphocyte proliferation assay because usually this experiment takes 2 or 3 days. I am afraid all B lymphocytes would be dead, limit references I found that involving this...

Wish for your reply, gratefully!

I use primary keratinocytes (KC) from the oral cavity, and experience that several factors can start early differentiation. Saurabh Mandal and Yakun Wu mentioned several factors:

- area (ours KC do well in T25 but not bigger flasks)

- number of cells per area (to few KC will start early differentiation)

- medium (to much calcium starts differentiation)

- splitting (never grow to confluence; check media used to split, return quick back to right medium; our KCs do well for 6-7 passages but start to differentiate after)

- age of the donor (we have bad experience with elderly donors)

- site of biopsy (KCs from some sites grow better then other sites)

Do you want to generate independent spheroids .

Varying concentration of serum with methyl cellulose

Thank you all for your suggestions. I've considered them all and I will most likely go with AAV transfection. The reason I want to stay away from electroporation is due to the low cell viability and my samples tend to be small.

Thank you very much Charles D Anderson

For passage experiment make sure the the media is in low calcium by using Chelated FBS. Hope this helps.

Regards

Have you gotten the cell line?

Timir Tripathi , certainly you are aware of the following article

¹Department of Chemistry, University of New Hampshire,

²Materials Science Program, University of New Hampshire

Original 'web location': =

for the following 2 URL's: after pasting the link-address into your browser, delete first the underline character " _ " in between "http _ and s" before you click on "RETURN":

[ http_s://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226083/ , for the original PDF:

http_s://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226083/pdf/jove-117-54803.pdf] , in the pdf:

cf.: 2. Isolating Chromatophore Pigment Granules and

3. Pigment Extraction

and BTW, there exists also a Video of the technique(s):

(NB: quote: "This is a sample clip. If you're new to JoVE sign up and start your free trial today to watch the full video! If your institution has an existing subscription, log in or sign up to access this video." End of quote.

There is an "old" source available (unfortunately only PPV, no warranty for "isolation techniques" contained) ):

Volume 3, 1969, Pages 307-353

(NB: same as above = delete first the underline character " _ " in between "http _ and s" before you click on "RETURN": applies to:

http_s://www.sciencedirect.com/science/article/pii/S1546509808601168)

In Vitro Cellular & Developmental Biology

International Review of Cytology

Volume 143, 1993, Pages 191-216, 216a, 216b, 217-255

(NB: same as above applies to:

http_s://www.sciencedirect.com/science/article/abs/pii/S0074769608618768 )

Fish chromatophores as cytosensors in a microscale device: detection of environmental toxins and bacterial pathogens.

(NB: same as above applies to:

http_s://www.ncbi.nlm.nih.gov/pubmed/11841070 )

P. Andreas Svensson and Helen Nilsson Sköld 2011,

Correct Citation: Andreas Svensson and Helen Nilsson Sköld (2011).

Skin Biopsies as Tools to Measure Fish Coloration and Colour Change, Skin Biopsy - Perspectives, Dr. Uday Khopkar (Ed.), ISBN: 978-953-307-290-6, InTech, Available from: http://www.intechopen.com/books/skin-biopsy-perspectives/skin-biopsies-as-tools-to-measure-fish-coloration-and-colour-chang

11. Effects of MSH on Pigment Cells https://www.karger.com/Article/Pdf/415282

...confessing / believing, it might be personal 'fortune' to get presented a "comprehensive / universal' protocol for isolation of chromophores ....

Best of luck, WHM

try cell-tak (https://www.fishersci.com/shop/products/corning-cell-tak-cell-tissue-adhesive-3/p-90828).

1) A study from Sigma on stability of insulin and IGF-1 in cell culture:

" Insulin is degraded in cell culture by enzymes (insulinases) secreted by cells into culture media. In addition, insulin is more rapidly internalized and degraded compared with IGF-I and LONG®R3IGF-I. The extended cell culture stability of LONG®R3IGF-I results in prolonged activity and associated benefits to cell culture. (Graph 5)."

In their conditions, 50% degradation of insulin during cell culture occured after 1.5 days.

2) I would supplement it further with trace elements like selenium as it was done in further versions of F12 media (this way the media will provide all the necessary nutrients/elements - though, the concentrations won't be necessarily optimum):

3) If you don't add any protein to the media (e.g. 0.1 mg/ml BSA or some more indifferent protein), there may be a problem with adsorption and inactivation of the tiny amounts of growth factors on plate walls. That's a real problem when you dilute enzymes - e.g. luciferases - to very low concentrations:

Cos7 and SH-SY5Y cells were used in my experiments. I found Lipo3000 is toxic when incubation overnight. So I change medium 4 hours after transfection, the protein expression is good. Hope it is helpful!

First I would recommend to keep right ratio of cells to Trizol reagent. For I mln of problematic cells such as neurons I would use minimum 1ml of Trizol. You can use double Trizol method. Isolate RNA by Trizol as usual, then air dried pellet treat with DNAse, then add another 1 ml of Trizol into DNAse/ RNa mixture and isolate RNA again w/o DNAse treatment. Also adding glycogen (see instructions for Trizol) into Trizol itself or into isopropanol helps with higher RNA yields. Make sure you never pick up interphase when collect aqueous phase after chloroform addition- better pick up less aqueous phase, then get DNA/proteins contamination. Also combination of Trizol/columns method will help a lot to get pure RNA. After addition of chloroform and collecting aqueous phase you need to add equal volume of 70% (important!, not 100%) ethanol to aqueaus phase and put mixture on RNeasy columns, then go through steps written in the kits protocol. Also if you have too many dead cells your A260/280 ratio will be much lower compare to healthy (>90%) viability cultures. So you can use Ficoll for example or dead-cell removal magnetic kits to get rid of dead cells. Good luck!

Hello Elisabeth,

Here is an example, where primary cultures of mouse ventricular cardiomyocytes were labeled by 2 μM EdU for 12 h:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4960126/

In general, it's a balance between EdU concentration and length of incubation time - higher EdU concentrations allow a shorter incubation time while lower EdU concentrations for a longer incubation time.

I suggest using a concentration of 10 µM for 1-2 hours as a starting point.

Do you want to do a pulse labelling experiment or monitor the synthesis of new DNA over a longer time?

If properly lysed, 1mio of any cells should give you roughly ~ 100 µg, so you should get to around 1mg/ml in protein assay, not 1000 times less.

So the basics: 1: You have enough cells, e.g. you see a clear cell pellet before adding lysis buffer? 2. If lysis works, your pellet after lysis centrifugation should be much smaller than the cell pellet. 3. Protein detection: Most assays do not detect such low amounts as 1 µg/ml....(e.g. lowest for BCA is 5µg/ml) You're sure there is no calculation error and you indeed already have 1mg/ml?

Best

Christian

It seems to me that it is more organ culture than primary cell culture.

Routine use of antibiotics in cell culture is not recommended. since , antibiotic resistant strains may develop and may cause resistant cryptic infections such as mycoplasma. Moreover, some antibiotics may have effects in cellular functions. Otherwise, primocin is a good choice.

We had similar problems using mouse embryonic neuron cultures. When aged, the cells do not appreciate to be dry (without medium) even for very short periods. Before treating those cells we change our medium by sequentially removing very gently (without any swirl) half of the volume contained in the well. After three or four operations we consider that the medium is now renewed and we add the substance to test, keeping one well as control. We prefer also glutamine to glutamax which appears to be toxic in some cases.

Do we still use such methods? It is too old method. Nowadays, you just have to use ultra low attachment flasks or nunclon sphera flaks and you can easily get sphere.

Hi, can the Adult brain dissociation kit be adapted for other animals. My interest is for macaque species

Hello Catherine,

Don't use Collagenase. You can try to use trypsin/EDTA to separate cells from the collagen gel. I am regularly using the trypsin/EDTA solution. It works perfectly well.

If you harvest during exponential growth clearly that may play a part in your interpretation of experimental results. However that was not the issue. All cells were of the same cell type to within a small number of spontaneous mutations perhaps. The only difference between the plates is that one plate contains hormone while the other does not. The inoculate for each paired plate comes from the same individual. I assume growth curves were obtained for cells with and without hormone so you know if hormone has an effect on cell growth and what if any that effect is so if such an effect exists you may consider it with the rest of the experimental results. Given all this unless some strange effect happens that requires explanation. This is statistically a paired t test. Any effect on the growth is known so any correction necessary can be made, such as was suggested by Dr. Ebert. Again when all necessary corrections, transformations and what have you are finished. The comparison is the paired t-test. This is exactly how we did chemical mutagenisis studies in Herman E. Brockman's lab many years ago. See the link for publications from some in the group with Materials and Methods sections. I apologize for my poor description but this was 50 years ago for me. Mea Culpa.

https://www.google.com/search?q=Herman+E+Brockman+Neurospora+yeast+mutagenisis&rlz=1C1CHBF_enUS847US847&oq=Herman+E+Brockman+Neurospora+yeast+mutagenisis&aqs=chrome..69i57j33.47880j0j8&sourceid=chrome&ie=UTF-8

Again, I see no reason why a poorly remembered something or other affects either the statistics or the biology. As always the assumptions of the test should be checked. Best, David Booth

Hi Laura, I found an interesting paper tht compares different approaches for isolating cells from brain tissue, maybe it can help you. You might want to try change your reagents.

Thank you John! I will look for UpCell dishes. Have a good day

I realise this question was posed and answered some time ago, but I would be interested on hearing some more on the topic - would anyone suggest adding RNAse inhibitor prior to sorting during cell isolation, or is it sufficient to add it after sorting the cells?

The majority of bacteria in fish are in the external mucous coat or the gut. So one approach is to minimize these microbial populations by changing fish husbandry prior to collection:

Hi Prasanth and Andreas,

I will provide more info on the experimental setting. So I am working on wild type mice and the neurons are first isolated and plated. Afterwards I am aiming to transfect them with 1) pX330 plasmids expressing cas9 and the gRNA for my specific target and 2) a selection vector harboring the sequence that the gRNA should recognize and cut. It also contains a frameshifted non-functional blasticidin resistance gene. When the cas9 cuts the sequence on the plasmid a frameshift will repair the blasticidin gene.

Before starting the experiment I want to find o