Im working with immortal PAM, I maintain at 37°C, with CO2 and I use recommended growing cell solution. (MEM). I change the 20 ml of solution twice time by week. I started from azote freeze cell and it's take 1 week to get confluent in T25. I used 2ml of trypsin (with EDTA) to switch it in T1. I will get 100% confluent surface to produce 24-well plate and make an infection.
My problem : A lot of big hole and cell cluster appear in the bottom of the flask. I'm still waiting 3 weeks but noting really change.
any suggestions please?
Thanks
Hi Annie,
There are several ways to ensure that your cells are seeded evenly and that there are not random bubbles or differences in cell density:
1. Make sure that there is plenty of media and that the flask is sitting level in the incubator. If the flask is not level, the cells will be uneven as they seed. The extra media helps prevent bubbles from adhering to the bottom of the flask (the usual culprit for random holes).
2. Make sure that your flasks or dishes are evenly coated with cell adhesive material. Some tech companies are better at this than others. Perhaps try a flask or dish from a neighboring lab for comparison.
3. Be sure that your cells are fully resuspended and well mixed before seeding. This cell clumps sound like cells that were not fully resuspended while adding.
Hope this helps and good luck.
Hi Annie,
There are several ways to ensure that your cells are seeded evenly and that there are not random bubbles or differences in cell density:
1. Make sure that there is plenty of media and that the flask is sitting level in the incubator. If the flask is not level, the cells will be uneven as they seed. The extra media helps prevent bubbles from adhering to the bottom of the flask (the usual culprit for random holes).
2. Make sure that your flasks or dishes are evenly coated with cell adhesive material. Some tech companies are better at this than others. Perhaps try a flask or dish from a neighboring lab for comparison.
3. Be sure that your cells are fully resuspended and well mixed before seeding. This cell clumps sound like cells that were not fully resuspended while adding.
Hope this helps and good luck.
Annie,
One more tip that I would suggest if you continue to have trouble with this is to resuspend the desired number of cells in a relatively small volume say 2mL then from this take the volume that will give the desired number of cells that you wish to seed and dilute that volume to the final volume you wish to add to the flask. So for a T75 you may want to suspend the cells in 2 mL of media by gently pipetting. Then take the volume that will yield the right number of cells (say that in this case it is 1.75 mL) and add 8.25 mL of fresh media to give 10 mL in a 15 mL falcon tube. In the falcon tube, mix the 10 mL thoroughly, then pipette all 10mL into the flask being sure to cover the entire growing surface with media and avoiding the formation of bubbles. When doing this it is useful to make a slightly larger volume of media/cell suspension than you ultimately wish to seed.
Try adding 1 ml of essential amino acids to the MEM for extra growth factors every time you seed. Also, once you have seeded, try not to make sudden/rigorous movements with your flasks as the first 30 minutes after seeding is really crucial so the cells can adhere. I usually move my flask slowly from the laminar flow to the incubator.
Hi Annie,
I'm just want to share what I've learnt before. When you add some media in cells after you've done trypsinization, try to pipette gently your cells by using small micropipette tips (usually 200uL tip) to separate the cells into single cells before you seed into new flask. So that,your cells will distributes evenly and will form monolayer (1 layer) in the flask.
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