Dear Khawla,

Usually Cell clumps after trypsinization is due to poor disassembly while pipeting them after you resuspend in the new culture media. After centrifugation and resuspension of the pellet, you can always pipet up and down, from 10 to 15 times for example, to guarantee that you will get nice single cells/lower amount of clumps.

Regarding viability, it really depends on cell nature, but if they have space to grow, it should not be a problem. Just avoid confluencies higher than 80%, and next time you tripsynize, do it more carefully.

Best regards,

Hello Khawla

Do not overexpose cells to trypsin. Generally, cells are exposed to trypsin for 3-4 mins. Overexposure to trypsin may be damaging as it may cause cell death. The DNA released from dead cells is responsible for forming clumps of cells and debris because DNA is sticky in nature. Also, as Lucas de Freitas Pedrosa has mentioned, avoid trypinizing overconfluent cultures.

If you follow these, culture should remain healthy.

Best.

Dear Lucas de Freitas Pedrosa

Thank you for the reply, I usually do as you mentioned but this time my undergrad students are doing their experiment and I wanted to check their work. moreover, I decided on a low amount to be passed into new flasks because the weekend was coming, so space between cells was good.

Thanks for the tips!!

Dear Malcolm Nobre

Thank you for the reply!

Honestly, this is what I am trying to avoid! exposing cells for a long time to trypsin, I usually incubate it for 3 minutes and pipet them until they are separated after adding complete DMEM, but this time my undergrad students are doing the work without me and I wanted to check their work and if it is fine to use them for transfection.

It's new to know that released DNA might cause clumps as well!

thanks for the tips!!