This could be due to overgrowth.

You may need to passage the cells more frequently and pasage the cells before they reach 90% confluence.

Also it may happen that certain areas are more dense compared to other areas so although total number of cells may be less than 90% confluence but the uneven distribution might create areas where cells are devoid of surface to adhere.

When cells don't get attachment surface they tend to stick to each other, which may explain cellular clumps. Cells are alive in clumps, however may interfere in your assays.

I agree with Sudeep, one simply solution we used to avoid the issue was to pre-coat the plate with 0.1% poly lysine and allow it to stay for 30 min and wash it of with DPBS and later plate the cells on the pre-coated plate. As 293T cells lift off easily, handling the plate gently is suggested.

Sudeep Kumar and Nagarjun Kasaraneni Thank you so much for your valuable suggestions. I think in my case the main reason was cell overgrowth, because i often passage when the confluence was around 90%, and the clump formation.

Dears, as my cell are very precious for me and don't have the stock. Is there any way to remove the floating cells without effecting the normal healthy cells ??

washing the cells with PBS and then changing the fresh medium will be helpful ?? or pls suggests me some other way, if there are ???

Thanks.

Regards.

Suggested ways to remove floating cells, mostly the floating cells are dead cells. You can remove the media completely and wash with PBS and add fresh media carefully.

To be more specific, you can trypsinize the cells and collect everything and centrifuge at 200xg for 5 min and most of the viable cells will be in the pellet and dead cells are in the supernatant (normally we use centrifugation to remove dead cells in suspension culture using the above suggested method). Hope this helps.

Maybe it is a late reply but I hope it helps others facing the same problem.

Most common reason for floating of cells in the media, is the delay in cells passages. you may didn't passage cells frequently before reaching 90% confluence, and you can avoid that by adjusting the cells concentration per the medium when you count them with hemocytometer.

So, most of floating cells are still alive and you can check this by taking 10uL of media including floating cells and check by hemocytometer.

If you would like to rescue these floating cells or recultivate them again, Just add 2 mL 0.25% Trypsin solution to your 10cm culture dish, incubate 2 min at 37 C then collect the whole solution and centrifuge for 5 minutes, then discard supernatent and suspend the pellet (which will contain only alive cells) in 10 mL new media and centrifuge again, discard the flow-through, and finally add 10 mL new media and count cells to adjust your cells concentration to avoid further aggregation or exceeding the adherence capacity and hence floating.