Do you know in which stage of the immunostaining protocol you are loosing your cells? Do you perform immunostaining in live or fixed cells?

Kazuhiro,

We have used either very clean glass cover slips or polylysine coated glass cover slips with adherent cells growing on them. We have experienced good adherence with mitotic cells through multiple treatments and staining procedures.

First, the cover slips must be very clean (straight from the manufacturer, they are not clean). They are washed in glassware detergent, rinsed, acid washed, and then rinsed, rinsed, rinsed in distilled water and finally in acetone before heat sterilizing (if cells will be grown on them). This is tedious, but any residual oil left on the CS will prevent strong attachment of cells.

We did not use machine washing or staining. Instead, we treated, fixed, stained, rinsed, etc by hand using CS held in self closing forceps and dipped in 25 cc beakers of buffer rinses. If there is strong agitation when micropipetting solutions onto CS or during rinses, mitotic cells may wash off. This is a somewhat delicate procedure requiring careful technique.

If you are still having problems with cleaned glass CS or slides, try polylysine.

Alternatively, a cytocentrifuge should make the mitotic cells adhere very tightly to clean glass or polylysine treated glass or plastic.

Hope this helps,

Robert Walter

Camilla Faoro

Dear Camilla,

Thank you. The cells on coverslips were fixed with 2% formaldehyde, permeabilized with TritonX100, and used for immunofluorescence. We guess we lost cells during this process.

Best wishes,

Kazu

Robert J Walter

Dear Robert,

Thank you for your good suggestion. We will use very clean coverslips that are washed well as you mentioned and poly-lysine coating. Thanks a lot for your help!

Best wishes,

Kazu

Dear Kazuhiro,

I usually fixed the cell as well in PFA and permeabilised them. I guess you loosing cells during the removing of the solution and washing steps. If your coverslip are in a 6-well plate, try to pipette in and out the solution at the corner of the well without touching the coverslip at all. For the permeabilisation and later steps I usually transfer the coverslip in a piece of parafilm on top of a small drop of the solution and let the coverslip "lay down" very carefully using tweezer to touch only a small corner. I had the same issue working with HEK293 cells cause they tend to detach quite easily, but never had issue with other cell lines (HeLa, mcf7, NT2, COS-1, Calu6, PC-3 for example) also without coating. I don't know if HCT116 tend to detach so easily as HEK293. It is also true that mitotic cells detach frequently. Do you let them recover a bit after nocadozole treatment? I usually combine a double thymidine block and nocadozole treatment for mitotic cells enrichment cause it is less toxic then using nocadozole for longer time. I can share my protocol if you are interested.

Camilla Faoro

Dear Camilla,

Thank you so much for your suggestion, which is very useful. We are also using the droplets method that you mentioned for washing and staining. We will try to enrich mitotic cells with thymidine block and nocadozole treatment. I would appreciate your help.

Best wishes,

Kazu

Please Go To this Link:

https://www.med.hku.hk/corefac/downloads/immunostaining%20protocol%20cell%20culture.pdf