Hi researchers,
I'm using Incucyte to do proliferation assay for HCT116. I seeded 3000 cells/well in 96-well plate. I've done this several times and the cells grew ok. But this time, the cell attachment is very slow. I upload the image in different time point after seeding.
What may be the reason for the slow attachment? The only difference in operation I can think of is that I mix more times before seeding, in order to make the cells distribute evenly in each well. Will this operation or "over suspend" do harm to the cells?
Thanks!
Oh the figure didn't show the time point. From top to bottom is 2h, 24h, 48h, 69h after seeding.
Dear Liu
The cells do not look like growing properly. I feel that some of the cells experienced mechanical stress due to over mixing, hence not growing properly. Another possible reason can be inappropriate passage number of your cells. Also, you should take into account the pH of the culture media if everything else is fine.
Try to figure out these possible trouble shoots.
gud luck
Hi Shuachen,
Your cells on the upper left and upper right look as if they are undergoing osmotic stress. Your buffer could be off or like what Sarita said the issue could be with your cells (passage limit, mechanical stress sensitivity etc.). Do you make your own media solution? Maybe you can test with a premade media if you can get a hold of one OR remake your media. Wishing you the best!
-ED
Hi Sarita,
Thanks. Next time I will try to mix smoothly. About the mccoy medium, I made it myself. One "strange" thing is the newly prepared medium looks a bit "orange" and over a period of time(about 1 month), it will look "purple". But this doesn't affect my cell culture for the passed few months.
The component are: 2.3g sodium bicarbonate+1 bottle Mccoy 5A powder(Sigma)+900 ml H2O+100ml FBS.
I think something does change during the storage of the medium but it doesn't affect my cell growth until now.
By the way, I use the same cells to do this proliferation assay and passage. The passaged ones still can grow.
Hi ED,
Thanks. I made my own medium but it worked fine for my previous experiments, even this time, besides proliferation assay, the same cells used for passage can grow. Maybe the medium store for too long? It's over one month.
Hi Shuaichen,
I already had this problem, and these are two issues you might also encounter :
- The cells you used for seeding were a bit much confluent in the flask of origin, so when you replated them, they already were in a contact-inhibition state. This happened to me with pancreatic cancer cells.
- Cells don't like plastic change, so try not to change the plastic while repeating the same experiment.
Hope it helps
Good luck.
Hi David,
Thanks for your sharing! Some of the cells are indeed a bit much confluent in the 6 well plate (I use 6 well plate to culture). I will pay attention to this later. But if it is the reason, will this inhibition last forever? You can see from the figures that even after 69 hours the cells still didn't attach properly.
After you notice the confluence problem and seed cells when they are not so confluent, have the problem be solved ever since?
Thanks!
Well, depends on how confluent your cells were before seeding, I guess they may even start to die yes... Actually this happened to me as well.
What i always do now is seeding while they are in the exponential phase, not too much confluent. Results so much better !
Hi Jun,
Do you mean during the storage of the medium, its pH decreased? Cause I've done this assay for several times and before this time I didn't see this phenomenon. The only difference I can think of is that the storage duration of the medium.
If it is so, any suggestions to avoid this? Because I see the every bottle of Mccoy medium I made experienced the same color change (light orange to purple over about 1 month). And what may cause this change?
Thanks!
Hi Sathyen,
I keep passaging for over 2 months. I also think about this issue and ask my lab member. But they say it's cancer cell, it will grow and keep the phenotype forever. Do you have any references that discuss the relationship between passage time and phenotype?
For trypsinization, I use 6 well plate to culture cells and put 0.5ml trypsin in each well for 3min at 37C. Then add 1ml medium to inhibit trypsin. Centrifuge at 1000rpm for 5min at 4C. Then withdraw the supernatant and resuspend the pellet in 1ml medium. Then continue the next steps.
Thanks!
Hi,
The color change of the culture medium is normal. It is caused by the interaction of the sodium bicarbonate in your medium with the (low concentration of) CO2 in the air. As the medium is becoming less in the bottle the color change is more intense. It means that the pH of the medium rises (purple means pH > 8), but there is no problem because when you put the cells in your CO2 incubator the high CO2 concentration reverts the pH to normal values (7-7.5). Unless the incubator is not providing adequate CO2.
Anyway, I think that the low growth rate of your cells is more probably effect of over-trypsinization due to the fact that they were confluent, as David pointed out. So, it is better to subculture the cells before becoming confluent.
Good luck with your experiments !
Hi Harris,
Thanks so much! I got a more comprehensive understanding from your answer.
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