5 replies to this topic

[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?Any suggestions would be greatly appreciated!

Thank you!
Chiara

[bob1]

There are 5 basic steps:
Blocking -use PBS or TBS with 3-5% BSA.  You can also use PBS-t or TBS-t as diluent
primary antibody incubation - work out apppropriate dilutions to use empirically.  Dilute in blocking solution
Washing -stringency wash in PBS-t/TBS-t 3-4 times for 5-10 min each
secondary antibody incuabtion - as for primary
washing - see above

[claire84]

can you explain me the role of the blocking solution?Is it use to prevent unspecific binding?What does PBS-t mean?Between each step I need to centrifuge, do you think I will loose too cells with this method?
I'm sorry, maybe these are dumb questions, but I'm not an expert...

[bob1]

The blocking solution is to prevent the anitbody binding to non-specfic sites on the cell.  It isn't so important for IF, but is for protocols such as western blotting.

PBS-t is phosphate buffered saline with 0.1-0.5% tween 20.

You will lose cells by centrifuging between each step, but most protocols will lose cells whether they are attached or not, so I wouldn't let that worry you too much.

[claire84]

Istead of BSA can I use a PBS buffer with FBS?
So for my experiment I should use PBS-t buffer with BSA (or FBS) and sodium azide, does it make sense?
Can someone recommend me a recipe for PBS buffer?

thank you!!

[bob1]

Correct, PBS with BSA or FBS will be fine. The azide is mostly there as a bacterio- and fungi-static.

There are many different formulations of PBS, usually they are Normal saline (0.9% or 150 mM NaCl), with phosphate buffering to pH 7.4.  Here is a basic recipe: http://www.qiagen.co...tLineId=1000177