Dear Noor,

Change in cells morphology due to starvation or any toxicant is inevitable. Cells respond time may be moderate to intense depending on stress level. Serum free media also poses mild stress. Culture of A549 cell is recommended in 10 % quality bovine serum. Moreover, that changes in morphology is different to different stress load and, thus, identifiable as apoptosis and necrosis. If sensitized to stress, adherent cells like A549 start to separate from the substratum of culture vessel and eventually become round and dead floating in media. Remember, you get also round but live cells after trypsinization of cells during culturing process.  

Hi Noor. Are your cells at the center more confluent? if so, I would recommend seeding your cells less confluent (40-60%) and starving with 0,1% FCS, and all the same other additives of the media in same concentration as full media. 

In my opinion, growing cancer cells for 6h in serum-free medium should not have any killing effect. The cell death might have occurred probably you are using higher concentration of lentivirus and/or transfection reagent.  I also experienced the same problem when I was transfecting breast cancer MDA-MB231 cells with siRNA, then I reduced the concentration of siRNA and transfection reagent and it worked fine. You can try this. All the best

dear mohd, 

before transduction, i seed my cell in 10% FBS. After i remove the medium containing serum, n perform transduction in serum-free medium. d cell show dying symptom. i think i need to do some assay to observe the cell characteristics in different percentage of serum

dear karen, 

the cell is just 50% confluent. but more cell at d corner compared to centre. the cells accumulate at d corner

dear sajid, 

i dont think because of lentivirus because i prepared one well for control (i dont put any virus inside). the cell inside control well also show dying symptom after 6 hours of starving

Dear Noor, i your lab we are expose to cells to 5 hrs of starvation (without serum and oxygen) to study ischemia-induceed cell death. So, I think 6 hrs of serum starvation also could be already harmful.

Is it possible that your cell line is too old, undergone too many passages and may not even be the cell line that you think it is because you might not have done STR profiling for a long time? If all these parameters are alright then it is a technical issue that may be solved by using a different CO2 incubator or a different cell line.

I  would look closely at the composition of the medium (check the pH and make sure the osmolality is not abnormal). Check also for bacterial contamination of the cell stocks and spent media; I believe your cells are sturdy enough to stand 6h hour exposure to serum free media, provided media  are well buffered to survive outside the CO2 incubator. 

best wishes

Michel R

 Hi Noor, are you sure those cells are dying? It seems the cells you see changing morphology are merely loosing adhesion or undergoing deadhesion. What happens if you take these cells and put them on full media again? Do they recover usual shape and state? If so, then I would not worry about that, as Mohd answered above. Anyway, it looks you are observing may be due to the surface of well bottom, which is not absolutely plain, so cells at the well centre are loosing attachment more rapidly. To test this try to use vitronectin or fibronectin recovered coverslips and see what happens. 

hi karen, the cells look like this

hi Stanislovas, i'm dealing with cancer cell line. i think cancer cell is resistant

hi Dibyendu, cell line that i used is just passage 5

Hi Noor,

What I think its a common phenomenon.

When you change the environment of a cell it reacts...!!! And cells have there own way. Many cells do many don't. Sometimes they start de-adhesion from the plate surface. And when they do they become more or less round. It just a minor shock to them and they regain their structure around 6h post change.

See when we change media the temperature changes even though we pre-warm it to 37C. And if we serum starve, some cells become round, but that does not means they are suppose to die. Its a quiescent stage they undergo, to reduce the cell-surface area. (Remember serum starve means 0.2% FBS)

I have a suggestion for Transduction: (If your cell line respond badly to serum starve): There is actually no need of serum free media for transduction. Because serum do not inhibit the process (like transfection with reagents), rather enhances...!!! Actually dividing cells uptake lentiviral particles more efficiently and that is possible in presence of serum. You should do it as follows:

1-~30% confluency is the best condition for transduction.

2- use minimum amount of media to cover the cells for 24h.

3-keep the virus particle for at least one doubling time (~24h).

4-use 10ug polybrene/mL of media. It helps in agglutination of particles and enhance membrane uptake.

5-replace fresh media at  ~24h post-transduction.

This may help you. Good Luck.