Hi, I had a lot of problems with this cell line. When I count the number of cells, I can't do it, because the cells form a lot of cell aggregations and i can't separate them. The cells was incubated with trypsin for 2 minutes, and the cells were detached of plate, but the cells form aggregations. So, i have a lot of problems with the MTT assay due to the number of cells is not very real. If somebody can help me... I dont' know if the problem could be the medium, because i was used DMEM instead of McCoy...and i don't know if could be this the problem.
Thanks!!
Hi, when you trypsinize, don't you see aggregation of cells? I grow this cell first in Mccoy's -5A but i saw that these cells grow in DMEM also, so I changed the culture medium because this medium is more used in my lab. So, do you think the medium is the problem to form aggregation? I'll probe Mccoy's - 5a. Finally one thing, the NEAA is very essential to avoid the aggregation?
Thanks Aeid Igbaria
The cell line can be maintained in both media mentioned above. Aggregation could be due to excess trypsinization of cells in the presence of EDTA. One can use TrypLE from Invitrogen for gentle detachment of cells.
For anyone in similar situation, aggregation could be due to trypsinization stage. Let the cells trypsinize for about 5 minutes (during this time, do not tap the flask to encourage detachment - tapping the cells to early during trypsinization is the most likely cause of aggregation). Also, you could mix the cell suspension by pipetting up and down to distribute them more evenly.
You could try trypsinizing them shorter; in my hands ~30 seconds is already enough to detach them and the longer I wait the more difficulty it becomes to resuspend them to single cells.
Hi Sergio Algar Lizana
You had asked this question before 3 years, I think you have all the practical points of HCT116, could you share your researches and advice.. Thanks
Hii when i used HCT116 in my lab its grow in DMEM easily, but when u perform trypsinization take less than 2mnt in incubator and keep it out than used hand=-shaker for aggregation of cells than centrifuge at 800 rpm.
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