I tried to subculture cells(hek293 cells) for the first time for a Cell Biology experiment, and here are the steps I followed:-

Cell subculture (*the media is a mix of 10:1:0.1 of DMEM:FBS:Pen/Strep)

  1. If cells are 70-80% confluency, please subculture the cells
  2. Remove media
  3. Add 5ml of PBS and wash the cells by tilting the culture dish few times
  4. Remove PBS
  5. Add 2ml of Trypsin/EDTA and mix well by tilting the culture dish few times
  6. Incubate the cells for ~2min at RT (until cells are detached)
  7. Add 5ml of media and resuspend well by pipetting
  8. Transfer 7ml of Cells/meida/Trypsin/EDTA into a 15ml of conical tube
  9. Centrifuge 300xg for 2min at RT
  10. Remove supernatant
  11. Resuspend cell pellet by tapping the tube 5 times.
  12. Add 5ml of media and resuspend by pipetting
  13. Add 10ml of media into a new 10cm culture dish
  14. Add 1ml of resuspended cells into the culture dish (containing 10ml of media) (1:5 dilution)
  15. Check the cells by a microscope
  16. Put the culture dish into 37oC, 5% CO2 humidified incubator
  17. Check the cells every day, and when cells are 80~90% confluency, do subculture.

I followed all the steps above to a T, and here is the cell culture under a microscope prior to culturing-

Pre-subculture cells

And the culture under a microscope post subculturing-

Cells post subculturing

I don't really know what the numerous black dots are in the post-subculture cell culture. I also can't quite understand if I can see any of the cells in the post-subculturing culture. So, I wanted some advice on what the black dots might be, and if the subculturing appears to have been performed correctly. Thank you in advance!

  • $\begingroup$ Welcome to the Biology Stack Exchange. Please take the tour and visit the help center for more information on this site and how it works. Please edit into your question which cell type this is, as that may help provide useful information on the effect you are seeing here. $\endgroup$
    – bob1
    Commented Mar 26 at 19:03
  • $\begingroup$ Thanks a lot for your warm welcome and your suggestions! I have edited the question accordingly. $\endgroup$ Commented Mar 27 at 5:05
  • $\begingroup$ How long after subculturing is the second picture taken? $\endgroup$
    – bob1
    Commented Mar 27 at 7:31
  • $\begingroup$ It was taken immediately after subculturing. $\endgroup$ Commented Mar 27 at 14:18
  • $\begingroup$ Were both images taken in similar conditions? The 'before subculturing' image on top looks like a dark-field image while the 'after' image below it looks to be bright-field $\endgroup$
    – timeskull
    Commented Mar 27 at 16:47

1 Answer 1


I'm thinking you are very new to cell culture - your first step should be to ask people in your lab.

You have a couple of problems here - it takes time for cells to settle and attach and start to spread out. Often several hours after seeding is needed for them to return to looking normal. Have a look at your cells now, as I've answered this ~24 h after you asked it. They should look more normal.

However, a lot of what you are seeing there is cellular debris. This indicates that you probably over treated your cells with trypsin and/or were very rough with your pipetting. It is normal to see some debris after seeding, but not too much.

In addition, your first photo shows massively over confluent cells, they are all tightly packed together and there are absolutely no gaps in the monolayer. This stresses the cells (even if they are not contact inhibited - HEK293 aren't contact inhibited), so they tend to attach more firmly than they should. This means that you end up treating them with trypsin for too long and then a lot of them die. You should be subculturing much earlier - there should still be about 20-30% of the culture area free of cells. The large, blobby areas in the top third of the first picture are not gaps in the monolayer - some of them look like senescent cells, and some are gaps left by dying/dead cells that haven't quite filled in yet.

HEK293 are a weakly attached cell line; in many instances you can tap the side of the flask firmly to detach them, you can even wash them off by pipetting. In my experience, you should only need a minute or less to detach them from cell culture vessel.

It might pay you to check the concentration of your trypsin/EDTA - often stocks are 10x or 100x rather than the 1x you need. Adding the wrong concentration will result in a lot of cell death as it is very hard to neutralize the trypsin.


You must log in to answer this question.

Not the answer you're looking for? Browse other questions tagged .