There are various kinds of HEK293(T) cells- cells that grow in suspension and cells that grow as a monolayer. Cells that attach well to cell culture plates are preferred. One can grow cells on poly-lysine coated glass coverslips for immunostaining. However, background is higher with poly-lysine coated glass coverslips. You may need to optimize staining procedure.

Compared to a cell line like HeLa, I've experienced massive amounts of cell sloughing in procedures that require washing when using HEK cells (e.g., immunostaining). You might try to allow the cells to adhere for 48 hours, instead of overnight. I've noticed a bit of improvement that way.

Also, it's crucial that you exchange fluids as gently as possible and ensure that you only add/remove from a side wall.

As Jordan suggested, gentle handling  helps but you can also try coating the plates with Poly Lysine. Here is the link to the protocol for coating , http://protocolsonline.com/recipes/stock-solutions/polylysine-coated-tissue-culture-surfaces/

Hope this helps!!!

Hello

i agree with all researchers , look at this protocol

Good Luck

http://www.its.caltech.edu/~bjorker/Protocols/Protocol%20for%20Immunostaining.pdf

Hi GuanQun,

1) As mentioned before, coating the slides would help. Ibidi sells different µ-Slides suitable for microscopy with different coating materials that you can choose from.

2) I think it is also important to keep cells around 70-80% confluent. They will detach even more easily if 100% confluent. Plating them for 2 days before the experiment will help too.

3) How do you fix your cells? Using fresh 4% PFA in PBS (stock: 16% methanol-free PFA) should work fine.

4) 0.05% Saponin is a gentler detergent for permeabilization compared to Triton as it preserves better some of intracellular membranous structures.

Best of luck!

Jackie

what about for confocal studies? Would coated plates affect confocal studies?

HEK293T Cells fix pretty well with 4% PFA and acetone but poorly in methanol or methanol:acetone ( 1:1)