Calcein-AM

From: Joël Jean-Mairet (jean@biotech.biol.ethz.ch)
Date: Thu Jan 07 1999 - 12:42:46 EST


Hi,

I am trying to set up an antibody dependent cellular cytotoxicity assay
using the well known calcein-AM. Cell loading is fine however I observe
around 50% spontaneous release.

1.) Are there any parameters I have to change in order to obtain less
spontaneous release? (for ex.  Amount of cells, loading time, dye
concentration)

2,) I am aware that lymphocytes can be separated from monocytes by plastic
adherence. Could someone give me a nice protocoll for this? How many
mononuclear cell should I put per cm^2 ?  Or, where could I obtain more
info on this protocol? I cannot find it because it is to "standard".

Thanx

joel



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