M. Kaur, L. Esau
pp. 119-126, (2015)
We have developed a simple, cost-effective, and labor-efficient two-step
protocol for preparing adherent cells for high-throughput flow cytometry.
Adherent cells were grown on microplates, detached with 2.9 mM EDTA (pH 6.14)
added directly to wells containing cell culture medium, stained, and then
analyzed on a flow cytometer. This protocol bypasses washing, centrifugation,
and transfer between plates, reducing the cell loss that occurs in standard
multistep protocols. The method has been validated using six adherent cell
lines, four commercially available dyes, and two antibodies; the results have
been confirmed using two different flow cytometry (FC) instruments. Our approach
has been used for estimating apoptosis, mitochondrial membrane potential,
reactive oxygen species, and autophagy in response to exposure to pure compounds
as well as plant and bacterial extracts.