It depends on the cell line you use. If it is a generic cell line with no special requirements, either glucose or glutamin will be the first component to run out. If you are experienced with that cell line you can approximate how long it takes for them to consume glucose in the media. It will also depend on the number of the cells you seed and the total volume of the media. I am guessing you are using a 96-well system (100 ul media) and from my expertise with several cell lines (10,000 cell/well) I would say that you would probably need to replace the media once in every 72 hours (longer for non-cancer cells). However the main problem is the drug you are testing in your assay. If it is a very stable molecule every time you replace the media concentration of the drug might increase. To avoid this people usually do colony formation assays (in 6-well, low number of cells, crystal violet staining, cheap) for longer incubation periods. If you know how long your drug will stay active in the cell, you can also add the drug accordingly. You may also introduce the drug at the beginning and only replace the media (without drugs) every 72 hours (or so), testing long term effects of the compound (instead of longer exposure to the drug).

It depends on the cell line you use. If it is a generic cell line with no special requirements, either glucose or glutamin will be the first component to run out. If you are experienced with that cell line you can approximate how long it takes for them to consume glucose in the media. It will also depend on the number of the cells you seed and the total volume of the media. I am guessing you are using a 96-well system (100 ul media) and from my expertise with several cell lines (10,000 cell/well) I would say that you would probably need to replace the media once in every 72 hours (longer for non-cancer cells). However the main problem is the drug you are testing in your assay. If it is a very stable molecule every time you replace the media concentration of the drug might increase. To avoid this people usually do colony formation assays (in 6-well, low number of cells, crystal violet staining, cheap) for longer incubation periods. If you know how long your drug will stay active in the cell, you can also add the drug accordingly. You may also introduce the drug at the beginning and only replace the media (without drugs) every 72 hours (or so), testing long term effects of the compound (instead of longer exposure to the drug).

 If you are looking for stimulated proliferation in your experimental group, then slightly starving the cells by not re-feeding may help you to see an effect.  If you are looking for inhibition of proliferation, then re-feeding will be necessary to promote normal high levels of growth in your controls.   For most cell types, refeeding must be done at least every four days to promote rapid proliferation. Without refeeding, proliferation will be diminished or stopped over seven days.

i am doing cell proliferation assay using 96 well plate. the cell is rat C6 cell line, cell number is 4000 cells/well.the culture is serum free media.

Dear Na Lu.

I have not worked with that particular cell line however since it is a fast-growing cancerous cell line I would say that they most definitely will starve in 7 days. 4000-5000 cells (with a doubling time of 20-24h) usually survive for 4-5 days easily without supplementation. By the way serum-free media may accelerate the starvation. 

Since you are using SF media, you can do multiple plates; For example without changing the media you can do cell proliferation assay for 2,4,6 day treatment.  In addition you can have two additional plates one change the media at 4 and 6 day to see if there is a change in  proliferation.  So their is a total of 5 plates (at least) for the first time experiment. Does it make sense?

In addition to Ali and Muthusamy, (adding on to Muthusamy's expt)

For cancer cell lines I would recommend the following: Using 96-well plates is going to be limiting.  In 96-well, the bigger problem is cells in control group becoming confluent, well before the others - if you are looking at inhbition.  At 5000 cells per well, you reach 40,000 cells per well by day 3.  This would be confluent for most cell lines in 96-well.

1.  When you say serum-free, you mean "synthetic" medium to allow growth without adding serum - serum-replacement media???  

2. You will need 12-well plates or 6-well plates (12-well is good to start with).  In 12-well plates starting with 2000 cells per well, and at least 2 mls of medium, there is enough surface area to go maximum 7-8 days before cells become confluent.

3.  Do the experiment that Muthusamy says, but with just untreated cells - at 2000, 4000 and 8000 cells per well (one plate is good for all), then check at 2, 4, 6, 8 days. 

4.  Draw graph of cells vs days - should be a straight line = cells growing evenly.

3.  If you do not keep the cells out too long, it is a good idea to replace medium on day 3.-4. Just suck off old media. then add new medium

** If you use drugs, also add drugs at your conc. at day 3-4.

** Also, if you do not keep the cells out too long, there will be enough of cell activity to replenish co-factors.  

** If you are worried about cell made co-factors, just add 2 mls medium +drugs to the existing medium and continue growing.

Hope this helps ! 

i think replacing the medium every 72 hrs is not a bad option provided you are supplementing them with the same amount of drug used initially on the cells.

The culture I used was F12K nutrient mixture(GIBCO), I have done the experiment for once and found cells didn't grow confluent.I found that the cells exposure to highest two concentration of my protein clumped together and died, and I could't obtain a dose response curve.

Ah. that may be the problem.  F12 is NOT a medium designed for high cell density - it is a cloning medium designed  for growing cells from extremely low densities.   You would probably need to change this medium every day to provide enough amino acids etc. for a rapidly growing line. 

You might want to try a medium with high levels of basic nutrients like DMEM, or people sometimes mix DMEM 50:50 with F12 to combine fairly high basic nutrients with the extra vitamins etc in F12 which partly help to replace serum.  You could replace this medium on day 3 and day 5.  As someone else said, it is also difficult to get high cell densities in serum-free medium.  Can't you add a little bit of serum, like 0.5%?

In the cell proliferation assay performed by Jiang and colleagues (PMID: 26400523), 5,000 cells of C6 glioblastoma cells per well of a 96-well plate are cultured for 4 days without any mention of refreshing the medium. This leads me to think that they did not refresh the medium. If you are using the same serum free medium used by Wolfe and colleagues (PMID: 7000795) then there shouldn't be a difference in the growth curve with vs. without serum. Moreover, Wolfe and colleagues mention that they change the medium on day 4 (figure 1).

Changing the medium every day is stressful on the cells and is likely not necessary while leaving them for an entire 7 days without changing the medium will lead to nutrient depletion. Judging by the literature I just cited, I would refresh on day 4 and this, according to data from Wolfe and colleagues should not affect cell growth.

I hope this helps.

Barae's comment "Changing the medium every day is stressful on the cells ..." is so very important but many people forget.  Changing the medium often (even in the 3-4 day schedule I suggest) is also stressful...but..

The concept that the cell medium needs to be refreshed comes from the concern that in complex serum, drugs can be bound up and "depleted" or can be degraded.  In this case, the balance between dug replenishment and growth stress has led to the 3-4 day regimen.

@Na Lu:  It looks like you may be doing assays in a primary cell or a primary-like cell line.  

1.  Firstly F-12K is not exactly the same as F-12.

2.  F-12 and the Kaighn's modification of F-12 (F-12K) were designed as BASAL media where (a).  serum can be inhibitory to some cells (b). AND/OR where one needed to study the effect of specfic growth factors, especially at concentrations that are far above what is available in serum.

3.  This comes from cloning of freshly isolated organ tissue where specific cell types from the organ/tissue were to be selected (like hepatocytes) - which were to be selected by their response to specific growth factors.  In this case, one wanted to suppress the growth of other cell types that would be stimulated by the mix of serum factors.

4.  The BASAL medium was formulated to compensate for the loss of some serum factors like amino acids, small molecules that appear to be generally required for these cell types.  Thus, it is labeled as a "Serum-free"  or "Reduced-serum", BUT it means that you must add the growth factor for that specific cell type.  Otherwise the cells will not grow. So you are removing serum because you want to add a specific growth factor.  It is "Nutrient-rich" in the sense that it provides EXTRA for the amino acids, and small molecules that would come from serum - but it does not necessarily imply that the cells will grow completely serum-free.

5.  I s this what you are doing?

6.  Cell clumping and death at high concentrations may just mean the protein you are adding has impurities.  In case of serum, it can actually protect from these small amounts of impurities by for e.g. albumin binding.

7.  If you are using concentrations of your protein higher than what you see in serum, adding serum at 0.1% - 0.5% may actually help buffering and binding impurities, but that you can only do by testing.

6b. It could also mean the protein is too stimulatory and the the absence of enough nutrients in the basal F-12K medium.  You can compensate for this by increasing the volume of F-12K 2-4 times (e.g 100 uL to 300uL for the same number of cells), OR adding non-essential amino acids, or other common supplements like amines mixtures, nucleosides etc. commercially available.

A long reply !! Good luck !!

what are you trying to do here- if its a tumour cell line other assays such as clonogenic assay may be better?

Thank you for all your valuable help!

@ Dr. Prem Subramaniam, you are exactly right, I am testing activity of BDNF and GDNF in Rat C6 cell line, BDNF is a brain-derived neurotrophic factor and GDNF is neurotrophic factors, so I need to remove serum.

 Next step, I will optimize experiment conditions for cell proliferition assay:1. change cell number and incubation time 2. 0.1%-0.5%FBS adding to the serum free medium 3.change the serum free medium(Wu Xf,et al,2008 used OptiMEM™ (Invitrogen) containing 0.5% BSA to do cell proliferation experiment. ), and to see if that will better.

For the long time and if you want to check what is the effect of your drug on cell proliferation, I would try: SEED CELLS IN PRESENCE OR ABSENCE OF DRUG AND TREAT THE CELLS AS YOU ARE DOING WITH normal  BACKUP FLASK (If you change media every 2-3 days and pass them once they are in confluency, try to do this for your experiment); just count cells passage by passage and check the viability by trypan blue stain for each step and seed again the untreated and treated cells by using the same number you will use to start the experiment. If the drug has effect on proliferation you must see it at certain time point.

1)longer exposure to the drug?

2)after you have answered question 1, then remove the drug at that point the compound will clearly show an eventual effect on cell proliferation to check THE LONG TERM EFFECTS OF THE DRUG

I agree with Prof. Subramaniam.

I avoid to change the media. 

Since media change is very stressful for cells and cell density is relatively low in this assay it is better to avoid media change. BUT 7 day is too much for l-glutamine hence try to use media with glutamax. L-alanyl-L-glutamine dipeptide will not degrade into ammonia during storage or incubation like L-glutamine.

No more than 3-4 days. .