In Vitro Cell Culture - Science method

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Dear Reza Ghahremani

Minimal plant tissue offers many advantages over big explants. In case of TCL, cells are directly in contact with the nutrients, and have maximum chances of organogenesis and somatic embryogenesis on optimized growth regulators.

Dayanna S. Costa Thank you

By control group, do you mean cells grown the same condition but without the cancer drug?

You need a no drug control under the same conditions as the drug but without drug. And yes the control cell must have high survival.

It is possible that extended serum starvation induces apoptosis in these cells. It is critical that you know the survival tendencies including plating density and resistance to serum starvation induced apoptosis.

Understand that serum "starvation" often refers to the time since fresh serum (media change).

Cells are supposed to die without serum. The more "normal" a cell line is, the more sensitive it is to serum starvation.

72hours with no fresh serum in many cell lines increases apoptosis without any other treatment.

I have tissue biopsy punches stored in trizol at -80. Can anyone share the protocol to isolate RNA from such samples. Is there a possibility to isolate DNA, RNA and protein in same sample with trizol?

I am currently doing a clonogenic assay on MCF-7 cell lines. Please how do I determine the volume of extract to add to my 6-well plate already containing 3 ml of my media+ cell suspension?

I need urgent answers cuz I need to add extract after 24 hrs of seeding the cells.

Thanks in anticipation of reasonable responses.

I understand this has already been stated; however, I wanted to recapitulate the advice of using freshly prepared reagent.

I've been performing the MTT assay on a variety of cells over many years, and my recent anecdotal experience, this discussion board, and some pub-med and google surfing led me to believe that the use of reagent which had been frozen could have been the source of the issue (hear me out!).

I prepared the reagent as instructed by the manufacturer (Thermo-Fisher) ~1-2 weeks ago, filtered, and made 10 mL aliquots to store -20*C wrapped in in aluminum foil. I thawed the reagent to use in my recent treatment time course (incubating MTT for 3 hours), but noticed there were some bright blue production among the purple formazan crystals.

I also noticed some precipitate which had crystalized onto the 15 mL conical tube, and stayed there regardless of light vortexing and even pipette scraping.

I've seen the blue production before, but as a PhD student, I did not think twice about it (my previous lab, MTT was always stored in -20*C).

All that said, I think that the preparation, storage, and care taken during use could contribute to the differences.

Although TF and other companys' protocols state that it may be stored for ~6 months in -20*C, I believe the freeze-thaw conditions could cause these differences. The colors produced by the formazan dyes will vary depending on the tetrazolium salt used as a substrate for the reaction; therefore, if the salt precipitates or alters during storage, it could consequently lead to altered outcomes.

I'm not saying that this is 100% your case, or even mine for that matter, I just wanted to provide a little insight from my perspective!

Thanks!

Hi Min, you can check a recent review like this

and the references therein. Best: Karoly

Dear Nanami, the following papers maybe helpfull to find answer to your question.

You can isolate bacteria from surfaces and than you can characterize their biofilm formation capabilities on different materials surfaces like stainless steel or polymer surfaces. There are both quantitative and qualitative methods to evaluate biofilm formation.

Try harvesting the cells 24-48 hours after transfection. Hope washed the cells three times 1X PBS or the buffer used before harvesting, check the pH. Also, use ice-cold buffer, and perform centrifugation at 4 degrees. Check the protein content in the supernatant.

Hi Robert,

I found that Wi38 is a human lung fibroblast line, not from mouse. It is mentioned in the article you attached and also in the Sigma Aldrich website from where I wanted to get it.

I observe ~48hrs doubling time and seed cell @ 10K per cm2 usually need to split every two to three days.

Hi

Hi Shawn Mcginley

I have also exactly the same problem (different color previously and now).

can you help me by telling me if there was any problem in the cell growth in culture done using this medium (faint red color medium)?

Sure, if you perform this method in any case via the same procedure (12 h at 4°C) the results you get should be comparable but may differ from other data published in different papers /(using different protocolls, procedures).

@ Luis Fuente Cevallos,

1. Make sure that when you sterilize the seeds with EtOH or/ bleach, you don't sterilize them too harsh, which can kill the seeds. Follow a good protocol from an good journal.

2. You can grow some seeds in soil to see whether they germinate or not.

It was based on a response curve for a active phase protein.

A digested samle.

looking for inflamatory profile.

IC50 for a compound/inhibitor for a cell line depends on several factors, including the cell line in question, solvent for the inhibitor, culture conditions, and proposed endpoint for the treatment. Therefore, it needs to be determined empirically under the desired conditions for the proposed endpoint (when known). If a study has used much higher concentration of the inhibitor for a similar endpoint, it could be due to due to other variables (cell line and culture conditions etc). It is always better to find out an optimum IC50 before performing an experiment.

Hi An, by definition, ECM proteins that are not soluble are integrated into the extracellular matrix.

It does not matter if this occurs in vivo or in vitro.

There could be many reasons for lack of in vitro CTL tumor killing:

HLA or H-2 mismatch

Leukemia cells may lack TAP

Down-regulation of MHC on tumor cells (check with labelled Ab)

CTL lack perforin or Granzyme B expression (check by Flow Cytometry)

Enhancement of Reproductive Heat Tolerance in Plants

April 2015PLoS ONE 10(4):e0122933

DOI: 10.1371/journal.pone.0122933

LicenseCC BY 4.0

John J BurkeJohn J BurkeJunping ChenJunping Chen

It seems to me that GABA and compounds which mimic it are toxic to neurons. You can try decreasing the dose of the compounds you are using.

However, it is quite difficult to exactly mimic culture conditions described in other papers. There are differences in chemicals, culture environment etc.

May be you can try primary culture of hippocampal neurons as described: Development of GABAergic Synapses in Cultured Hippocampal Neurons

J Comp Neurol. 2006 Apr 10; 495(5): 497–510.

Catherine Croft Swanwick, Namita R. Murthy, Zakaria Mtchedlishvili, Werner Sieghart, and Jaideep Kapur

Regards

There are two kinds of starvation: one is serum starvation, where your cells will slowly arrest their proliferation (block in G1) for absence of growth factors provided with FBS, they will remain vital for some time but, depending on the cell line, they will eventually start to die. You should be able to obtain a good starvation in 24-48 hrs with FBS free medium. Another kind of starvation is fasting, so glucose-starvation. That is more complex to carry on over time, since your cells will not have any energetic substrate to use, will suspend their metabolism, and start to die quicker as glucose reserves run out (they could also undergo autophagy to survive and take time, but not for long). Anyway, cell cycle arrest in G1, observable with flow cytometry is probably the best way to confirm effective starvation. I don't know if there is a way to get a chronic starvation in cells, for sure is possible in animals, but I have some doubts about the possibility to keep starved cells alive for more than 48-72hrs.

If this does not answer to your question, you could provide additional details about what you need to do.

Good Luck

Hi Danielle,

I used this protocol and it worked very well for me.

It also depends on how you thaw them!

Into warm medium, work quick, consider adding DNAase which always improves!

Especially if you need many cells and viable!

Why are my SW620 cells even growing at cold 4 degree temperature? M using this to be a negative control.

It's been long, and no one else answered, please excuse me for talking to myself.

I ended up dissolving DFO in ddH2O, and put it through 0.22um syringe filter to make it sterile.

The 100mM stock was aliquoted in black micro tubes and sealed with parafilm, frozen in -20C frig. To avoid repeated freeze-thaw cycles, I aliquoted it in very small volume, so that I can use it up within 2nd thawing. In this way, it worked just fine.

Hope this information could be useful to someone :)

Unless you find multiple studies that made use of ketoconazole as antifungal for cell culture treatments, I advise you not to use it. Ketoconazole has been documented to antagonize GRs (glucocorticoid receptors) and if your aim is to maintain the integrity of the cells after an antifungal treatment, your drug of choice might not be appropriate.

Check if this supplement might be of lower price that Amphotericin: http://www.invivogen.com/fungin

I think that no one will help in this, the whole package is one gram and it is more than expensive. You have to buy or to share with others and also buy. All the best in your work.

Dear,

There is no such formula to extrapolate an in vitro dose to in vivo studies. But you can opt to any of the following

1. If your molecule is completely new, then go for LD50 studies first to estimate the maximum dose which is safe to use. Then select any three doses below LD50 (I recommend you to go below LD25) and proceed for your pharmacological studies.

2. If you know the chemical class of the drug and its therapeutic area, then you can refer to any reported drug molecule in the same class for its dose and route of administration.

3. If you have synthesized new molecules by changing functional groups in any existing molecule (Refer point 2)

4. You can go for Pharmacokinetic and toxicity studies but it will consume lot of time. Again, if you know the chemical class of the molecule you can at least get an idea of the kinetics and toxicities of your molecule.

Hope this will be helpful. have a great day.

Dear all,

I now use the following protocol:

I take out 100 µL medium from each well (neurons are cultured in 24-well with 500 µL medium) and use this to make a pre-dilution of 14 µM AraC. Then I pipette 100 µL from this solution back in each well. This is how I achieve a final concentration of 2.8 µM in the well (more or less because there might have been some evaporation). I add the AraC at DIV3. At DIV8 I change the medium.

Dear Hanen,

Do you remove your test compounds from the cells before adding the MTT reagent?

Some compounds can react with the MTT and thus give false signals...

So, I would recommend to change the medium after the incubation time with the compounds and add the MTT in fresh medium or buffer to avoid a reaction with the test compounds...

Good look

Thank you Chunlin Huang sir!

Dear Fu Jiayao,

Many thanks for your answer.

I have made 3 days ago an overnight incubation in RPMI (#1640) without serum and glucose, but with amino acids, etc. In sum, the Hela cells in starved condition detached from the culture dish, at least in my hands, therefore, I am not sure if they would survive a longer period.

I have made an overnight incubation with Hela and MEF cells in DMEM low glucose and with amino acids last night, and the cells were fine. I think until Saturday I will have an idea if this protocol worked.

See the following review paper: The Combined Application of the Caco-2 Cell Bioassay Coupled with In Vivo (Gallus gallus) Feeding Trial Represents an Effective Approach to Predicting Fe Bioavailability in Humans. Link:

You can dilute the collagen during the gel preparation procedure; often the collagen stock solution comes with a protocol for this where you plug in the desired final gel concentration and it gives the respective amounts of sodium hydroxide, water, and PBS you should add prior to incubation. 

 What Charles said. In your case I would first try thawing the frozen cells on ice (it will take a long time but since you don't care about long term cell viability it shouldn't be a problem) wash (add and then centrifuge 0C-4C 10 minutes 500-1000 rpm) the cells with ice cold serum free cell culture media once (maybe let them sit in this for a bit after first centrifugation)  then at least 3 times with ice-cold PBS to remove the dead cells, freezing media, and regular media components. For extra protection against degradation I recommend the Cell signaling technology lysis buffer with roche complete protease and phosphatase inhibitors for your cell lysis buffer cocktail. 

Thanks Robert, that was very helpful! We have since checked in-house expression data for a-tubulin and found that our data corroborates the reference you cited - tuba1a (a-tubulin) expression fluctuates throughout myoblast differentiation. We will give GAPDH a try and move onto DJ-1 if needed.

Hi Ali,

In order to accept the impossibility of pipetting of the pure acetaldehyde, please look thru specifications of acetaldehyde, which has a density of  0.7850g/mL, packaging in AcroSeal™ Glass Bottle in Tin Can, and containing water 0.02% Max.

Best wishes,

Ilya 

Thanx alot for your help

Hi, I think that 8 year of storage are probably too much for every enzyme, also for trypsin that is a quite "highlander" enzyme.

I don't recomend to use it for your cells.

Dear Natalia,

You should consider the state of H2O2. Is the most important thing, before performing the concentrations and times. best regards

may be you should be doing a control test like seeding cells in a 60 mm or 30 mm dish and treat with very low concentration of your drug to visually check whether it is cell compatible or not. Before that you have to ensure in which solvent your drug is soluble. Then you can go for basic cell functional assay. Use the concentrations if reported or nearby concentration with different time points to check its IC50 on your interest of cell line

thanks Allende, we are using penicillin G and Streptomycin sulphate

Ok, so you want to seed 5 wells at each confluence. Let's calculate for 7, then. Generally, prepare your suspensions in a tube off of the plate first, then seed the resuspension onto the plate at the final dilution. (Not cells first, then medium).

Ok, so 7 wells at 200 uL per well will be 1.4 mL. This will be the final volume of your suspension before seeding. You can do this in a 2 mL tube with comfortable space.

7 wells at 12,000 cells per well is 84,000 cells. This is the number of cells from your concentrated original stock to the 2 mL tube.

You have 2,400,000 cells in 10 mL, which comes out to 240,000 cells per mL. By simple division this is (84,000 cells) / (240,000 cells/mL) = 0.350. The units cancel to mL, or 0.35 mL, which is 350 uL.

So, to get 84,000 cells, which is the number of cells needed if you evenly distribute your suspension across 7 wells for a final of 12,000 cells per well you need to dilute up 350 uL of cells from your original stock.

We know you need 1.4 mL of final suspension, so 1.4 mL - 350 uL = 1050 uL of medium to adjust the volume.

Take a 2 mL tube:

Rinse and repeat for all concentrations you want.

Hi, from my little experience working with MTT, I have observed that sometimes over-confluence of the well plate could account for  your observation. Also ensure that you are seeding the same number of cells per well in the treatment group as you have in the control. Then some  literature search on your extracts and the particular cell line you are using could also give you a clue. All the best.

Dear Edigar,

You are observing nothing unusual. Actually live cells via their enzymatic activity are able to reduce yellow MTT to blue formasan salts while dead cells can not. This is the basis of using this salt for measuring cell viability. I would recommend to use final concentration of MTT in plate at 0.5 mg/ml dissolved in phenol free incomplete media and filtered with 0.22 um pore size membrane.

Dear Isarin Thanaboonyawat,

First of all you should collect your cells in a 10 ml PBS after trypsinization or scrapping and then centrifuge to pellet down the cells. Before centrifugation mix cells well and count the cells using hemocytometer. Now carefully remove PBS without disturbing cells. Now you can lyse your cells by adding 0.2 or 0.3 ml pure water (may contain 0.1% triton 100X)  and again centrifuge at 10000 rpm and use supernatant for assaying SOD or catalase or protein estimation. 

Dear Gayatri Rajenda Kachh,

You can use fish cell culture media available from Sigma or many other manufacturer. For cell analytical purposes, you can use PBS, HBSS where cell intactness is required or water if cell lysis required.

@all ....many thanks for your guidance.

can i do transfection twice??  like usually we incubate for 48hrs after transfection, if i transfect it again after 24 hrs then would it affect the results? like it would improve the gene knock down or not?

Hello Nur,

The problem you are dealing with is most common during co-culture experiments. Since tissue scaffold's one side can't be isolated from other so using different media for separate sides is practically impossible.  Ideally you should acclimatize your both cell types to single media which is totally possible or you should try a different media like a-MEM in which both cells show optimum growth. Still if your cells are very specific to grow in these two media then you can try RAFT MEM maintainance media from Lonza. That is suitable to culture most of the cells for co-culture experiments. Rest you can ask the company people.

Disclaimer: I am not intended to promote any company product and the answer provided here is solely based on my personal experience.

Hope it helps!

This is known as the edge effect and it happend in any tyoe of plate. It is related with  with different temperatures between peripheral wells and  central wells and with the evaporation that the peripheral  wells suffer. One solution that  helps is to preheat the plates for 15-20 mins in the incubator before seeding the cells, these helps to maintain an homogeneous temperature along the wells. As you mention for 96-well plates most people avoid peripheral wells  and fill them with PBS or medium to maintain humidity and an homogenous temperature along the other wells. If you need to have  6  experimental wells I suggest you to use 24-well plates instead of 6-well plates, preheat the the plates in the incubator before seeding the cells, and fill the peripheral wells with PBS (same volume that you uses of medium and cells) and use the remaining 8 central wells for your experiments. I hope you find this information useful 

DMSO

Hi Filip,

In my lab, SH-SY5Y cells are differentiated by dibutyryl cAMP and RA for 48h. After that, those cells have pretty stable neuronal phenotype expression. So, we culture them for next even 48 h without differentiating media.

For me, yours protocol seems to be fine. So, go with that and Good luck!

hi Adam Yes the M smegmatis used in vivo is resistant to the antibiotics used in the plates. As I have transformed it with the vector bearing antibiotic resistance.

Follow up with the question, I did another experiment where I incubate the cells with this molecule but did NOT wash it away but directly fix and stain it with phalloidin. From what I saw, the cells don't have their characteristic actin rich dots in them anymore. Usually my cells have a lot of these dots, but these dots just disappear in these GM6001 treated cells. It's really weird. 

You're going in the wrong direction.  Increasing the concentration of compound in DMSO means using less volume in your cell culture - this DECREASES the amount of DMSO around, and thus minimizes the solubility of the molecule.  Similarly, filtering is simply removing compound from your solution, thus reducing the concentration beyond what you calculate.

What you need to do is figure out the LARGEST amount of DMSO your cells will tolerate, and then use the most DILUTE stock solution you can to supplement your cell culture media.

As for adjusting the pH: it might make it more soluble in DMSO, but you're adding it to culture media, which should be well buffered, and the little bit of acid or base in your DMSO should effectively cease to matter.  And that is, again, your problem: your compound isn't soluble in culture media.  Optimizing solubility in the DMSO stock isn't what you need to worry about.

As a further note: these compounds have been used in the literature a fair bit, on cell cultures.  You might just look at what other researchers have done.  And if that fails, why not just make the sodium salt of your compound?  Then you might not even need DMSO, as the salt should be relatively water soluble all on its own.

You must check the amount of sodium pyruvate in RMPI medium, because A549 cells are very sensitive to the low level of it. If there is less sodium pyruvate in RPMI than in the DMEM medium, add the missing amount of that compund.

Hi Sena,

I always use Media: FBS: DMSO as 50: 40: 10 proportion and it works for every cell lines I have used.

Cheers

Thank you so much Alan D Brooks for your detailed answer. It helps me alot

Hi Shilpa. I use inserts (Corning, polycarbonate and PET) to grow my cells - grows fine in both. There are various reasons for your situation:

(1) Have you checked if your cells need some coating material on the inserts? Eg. collagen, matrigel, fibronectin? 

(2) How many control inserts did you perform? If more than one, did all the replicates also show the cells dying on the inserts? If just one insert, try again one more time to see if it happens again. The cells probably just decided to die on your first experiment. My supervisor always tell me to do TLC (Tender Loving Care) on my cells. 

All the best!

Extract only works on some cells, but not all. Just like some cytotoxicity assay, some of the plant extract can suppress the growth of certain cell lines, but not all, because the mechanism inside the cells are different. 

Or you can try not using FBS when u want to incubate the cells with the extract, starvation allows the cells uptake more extracts. :) 

Thank you for your kind reply Maria

In my experience the likely problem for spot background is almost always with washing errors, especially the last step prior adding substrate. Depending on your method of washing, you could try to increase washing volume and times (5x vs 3x). In larger screens I usually opted to leave the very last washing step for longer period.

As others pointed out, blocking is key, and the more important if you shake your plates.

Are you able to culture other types, e.g. cell lines?  I wonder if perhaps the primary astrocytes are very sensitive to atmospheric oxygen concentrations (about 21%).  I suspect that they experience a much lower O2 partial pressure in vivo (about 3-5% perhaps) and possibly they can't keep up with the oxidative stress that atmospheric O2 puts on them.  I culture primary cells as well, and find that if I put them in 21% O2, the media changes to yellow almost immediately (no metabolites are produced that quickly) and the cells grow, just not very well.  Culturing in 3-5% O2 gets rid of that problem in my experience.  Perhaps you have a tri-gas incubator set up in your lab already -- if not, Airgas does offer 5% O2, 5% CO2, 90% N2 mixes for about $140 if I recall correctly.  You'd have to hook that up to an airtight container within your incubator.  But if it makes your cells grow well, it'd be worth it.

Dear Lakunle,

1) 2g/L glucose should be kind of standard formulation. If not, get lower strength and add your own, then filter sterilize.

2) 0.2% BSA means 2g/L. Proceed as above. Only makes sense when there is no serum present, of course (which will bring lots of own BSA)

3) I'd use a small pipet tip attached to a vacuum source. If your cells can stand it, just flip the medium out like when you'd wash an ELISA plate. If it is a short term experiment, you may do this directly at the sink, as strong sterility is not required anymore.

And wash the plate once or twice with the new medium in order  to remove traces of the old one. If medium should be too expensive, use PBS, ideally with an appropriate concentration of glucose.

Hi

You can try the protocol published earlier using LoVo cells in clonogenic assay-

Best of luck

The ability of the various trypsin inhibitors to prevent trypsin hydrolysis of benzoyl-L-arginine (BAPNA ) ethyl ester is measured spectrophotometrically 410 nm.

Thank you Dr. Ali Abdulmajid for the suggestion its a good book and will definitely be helpful in vivo study, but I want to know if there is any way for in-vitro model 

The protocol from cell bio mentioned if i use a clear black bottom plate lysing is not necessary. http://www.cellbiolabs.com/in-vitro-ros-rns-assay this is the kit that i am utilizing. 

You are welcome.

Positive control... well yes if you want to assess your current method, but maybe good to consider the Viromer RED as your main taxi.

Anyway, I renew my proposal, you are very welcome whenever you want.

Good luck.

Hi Paul,

For cultured neurons I would always go with lentiviruses. Calcium Phosphate has never worked so good for me in any cell culture.

Best,

Milica

10

It seems like that

Roche has excellent customer support center in Mannheim, many specialists speaking several languages, just call them during working hours, they can assist you in real-time with your application, troubleshoot. If there is a defective product lot, you can inquire replacement.

Tel verde 0800 80 66 80

Or write them an email with your problem at mannheim.csc@roche.com  

It may depends upon the mechanism of action of the drugs tested and upon the parameter studied. For an oxidative shock for example, 2-3 hours may be sufficient to observe cell death, if you want to study an effect on cell metabolism you have to wait until 6-12 hours at least and sometimes 24h, if you follow an effect on cell proliferation and growth, you have to follow your cell number during 24-48  hours, to detect apoptosis, it may takes 24h to 48 hours. I guess that for each compound of interest you have to determine a dose response-curve and a time-course.

Sincerely your's

D Duval Ph.D. 

Thank very much for your help. Do you know if there is a maximum percent of PBS in the media that would not interfere with cell viability? Thank you again to both of you. 

Best regards. 

@Marcela, I have done Mycoalert test (Kit from Lonza) for my cell line samples and media. It came out negative. Can I trust the test and proceed with my cells?  

Hi both 

Thanks for your comments the amount of TGF-b is  2ug.