Cell staining for fluorometric assay

[Claire84]

I'm trying to develop a fluorometric assay to monitor the expression of a surface protein in HL-60 cells, using fluorescence antibody. We would like to use a fluorescence plate reader to measure the fluorescence of antibodies bound to cells. Could you recommend me some protocol to stain cells plated in a 96-well microplate?Do you think I can adapt a protocol for flow cytometry?

Any suggestions would be greatly appreciated!

Thank you!
Chiara

[relaxin]

I do not have any experience in this type of assay, but I think you can adapt a cell-based ELISA protocol:

http://www.biotechniques.com/Posters/Ce ... 03227.html

Since the protein of interest is on cell surface, you can skip the permeabilization step. Fixing the cells is important, or you will lose some cells during the washing steps.

Flow cytometry will be too time consuming, if you have a large number of samples.

[Claire84]

Do you think centrifugation step, with subsequent removal of supernatant, is not suffiecient to avoid the loss of cells?

[relaxin]

It will be difficult to avoid losing cells, if they are non-adherent. Here is a protocol for non-adherent cells:

http://www.mitosciences.com/PDF/in-cell ... otocol.pdf

Disclaimer: I do not own any Mitosciences stock.

[CrowSan]

Perhaps rather than staining in the 96 well plate you stain in i.e. a 1.5ml eppendorf then aliquot set amounts of stainined cells (after labelling, secondary and washes etc) into the 96 well plate. Prior to adding to the 96 well plate do a cell count to account for any losses during the labelling procedure.

[CrowSan]

Oh one more thing if you are fixing cells (e.g. 4% PFA 10-15 min) then be aware that some non-adherent cells become very "sticky" to plasticware after fixation (I had a terrible problem with L1210 cells with this. Every time I transfered to another tube or pipetted I lost 10%. This may not sound much but imagine fixing, washing a few times, transfering to a new tube etc it soon mounts up to ~40-50% cell loss or more). If you notice this happening then try adding a small amount of BSA (0.1% or there-abouts)