Subculture the cells. Hope the following method would help. Good luck.

VI.1.4. Initiation of cell culture

A cell culture was established according to the method of Freshney (1987).  The cell culture was initiated by seeding a secondary cell 3T3 mouse fibroblast in a culture flask (Life Technologies Ltd. Paisley, Scotland; 25 ml) containing 5 ml media.  The cells in the flask were then incubated in an incubator at 36.5oC with 5% CO2 and 95% O2 gas phase.  After 48 h of incubation the cell line was maintained by subculturing on the same medium.

VI.1.5. Subculture

The cells were subculture by withdrawing medium from the flask and washing carefully (by manually shaking the culture flask) with Dulbecco's phosphate buffered saline (PBS) solution (5 ml).  Trypsin solution (3 ml) was added into the flask and incubated at 36oC for 10 min until the cells rounded up.  The cells suspension (3 ml) were transferred immediately into two tubes (10 ml) each tube containing 5 ml standard media.  The tubes were centrifuged at 850*g at 4oC for 11 min.  The supernatant was drained off carefully and the cell residues which usually stuck to the bottom of the tubes were mixed thoroughly with 5 ml fresh standard media.  The cells were transferred into a new culture flask and incubated at 36.5oC as described in section V1.1.4. above.

VI.1.6. Cell counting

To examine the effect of gossypol on the growth and multiplication of cells, four treatments cells were chosen namely To = cells + media, Te = cells + ethanol, TgL = cells + 5 mM gossypol and TgH = cells + 20 mM gossypol.  There were four replicates in each treatment.  The growth of cells was measured by harvesting at 0, 24, 48 and 72 h of incubation and counting the number of cells using a counting chamber (haemocytometer, Weber Scientific International Ltd., Sussex-England).  The cells were transferred into tubes containing 5 ml of a standard medium and centrifuged at 197*g for 4 min at 4oC.  The supernatant was decanted off and the cells were resuspended and mixed thoroughly with 1 ml fresh medium.  Using a micropippete, 20 ml of the cell suspension were transferred immediately to the edge of the haemocytometer chamber.  The pipette was reloaded and the second chamber was filled.  The cells were then counted under a light microscope.  Each chamber consists of 25 squares; each square (4 x 4 mm) was made up of 16 small squares of 1 mm2 area.  The cells were counted within the 1 mm2 area; and 5 replicate 1 mm2 areas were counted for each observation.  Since the area and the depth were 0.0025 mm2 and 0.1 mm, respectively, the volume was 0.00025 mm3.  Hence 1 ml (=1000 mm3) has the same volume as to 4 x 106 squares, and the number of cells counted in 1 mm2 had to be multiplied by 4 x 106 to calculate the number of cells per ml.

Thank you very much. 

Hi Kamlesh, have you figured out the reason for slow growth of MG63? I am currently working with MG63 using RPMI 1640 + 5% FBS and having a similar problem like yours. Cells seemed to stop growing or grow very slowly after the first two passages with a lot of floating dead cells. No mycoplasma. Really appreciate for your time! Thanks!!

Connie Long , I have worked only few times with MG63 cells. Currently, I do not work with MG63 cells. As far as I could remember, they need dense condition for the growth. So I will recommend to grow cells in high confluence condition. Thats the only advice I can give you at the moment.

Kamlesh Ganesh Pawar Thank you for your advice!!

Hi Kamlesh,I am currently working with MG63 and I grow them in EMEM media +10% FBS. I ensure that they are fully confluent before I split them, otherwise they seem to stop dividing well. I usually split them 1 in 3 (from a fully confluent 100mm dish) twice a week or 1 in 5 to grow over the weekend and they seem to be perfectly fine.

Thanks

Hi all,

I am currently working with MG63 and i am going to use DMEM +10%FBS+1%PSN. I want know the media should be in High or low Glucose.

Thanking you

Regards

VC

Hi, I guess your not fallowing any antibiotic. If so you can add 1%PS or PSG.

this may help to avoid contamination.

And also have problem in medium filtration, if filtration is not good then definitely getting contamination.

Thanks

Regards

VC

I recommend the composition of these MEDIUM" DMEM/EMEM +10%FBS+ L-gluatime +1%PS or AA "

So basically I don't get contamination in my culture media but I don't understand why my cells are dying within 2days. My media composition is DMEM low glucose+10%FBS+1% antibiotic. They grow properly for 2 days reaching a conflency of around 50-60% but then they start getting detached.