Please help me with this.
I'm working on cell viability assay. I used to seed the cells a day before adding the drug. My observations are: few cells are coming out while aspirating the old media.
1. Few cells are coming out while aspirating the old media. But this is negligible.
2. But while adding the fresh media containing my drug, the cells are detaching from the surface and even sometimes it forms a scaffold. I use multi-channel pipette to do the same and I tried manually changing media (slowly) using single-channel pipette as well. In both the cases, the cells were dislodged (approx. 60%).
I'm using HEK293 cells.
Many thanks,
Arun.
Hello Arun, try this may be this will be helpful for you
1. Seed the cells for 24 h so that the cells adhere properly.
2. Add the fresh media containing the drug by touching the wall of the plate by multi channel or single channel pipette.
Hello Arun, try this may be this will be helpful for you
1. Seed the cells for 24 h so that the cells adhere properly.
2. Add the fresh media containing the drug by touching the wall of the plate by multi channel or single channel pipette.
When I use HEK293 cells I coat the plate with Poly-L-Lysine.
1 well of a 6 well plate would require 0.5ml of poly-l-lysine at 0.1mg/ml. Remove and wash the wells in PBS then leave for a few hours to dry.
I would also use the poly-l-lysine several times before discarding it.
I could then use 500,000 cells/well of a six well plate for several days without loss of cells.
Thank you for the above suggestions
@Vijay Kumar: I can't incubate for 24 h because my drug becomes less effective when I leave it for 24 h. I added very slowly by touching the wall but still, the cells were dislodged.
@Raj Kumar: I use the flat bottom. I'm in the process of optimizing cell density now. I will update once I confirmed it.
@Eliot: This is one such suggestion when I searching for the solution. I heard that HEK293 will not adhere to the poly-L-Lysine coated plate. Anyway, if nothing works I will try this at the end.
Hi,
Please check again your cell culture plate type. Is that cell culture treated multi-well plate or non-treated plate? Please read following pages. Hope your problem will be solved soon.
https://www.researchgate.net/post/What_problem_may_non_treated_tissue_culture_well_plates_have_when_using_for_cell_culture
https://www.thermofisher.com/jp/ja/home/life-science/cell-culture/cell-culture-plastics/cell-culture-plates.html
Hello Arun,
HEK-293 are very sensitive to lower temperatures than 37°C. They detach from substrate very rapidly, when the bottom of the wells (and the medium) becomes cool. Ensure warm temperatures in your media and on the surfaces you put the multiwell culture plates on. And give new medium immediately upon removing old medium.
With HEK 293 Cells, less density will provide you a better attachment for the cells and avoid a scaffold detachment. 10.000 cells / wells will usually behave like that. I found 2500-5000 cells/well is better.
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