Two-step protocol for preparing adherent cells for high-throughput flow cytometry

BioTechniques
Mandeep Kaur, Luke Esau

Abstract

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.

References

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Citations

Apr 6, 2017·Cytometry. Part B, Clinical Cytometry·Chris P VerschoorCynthia Balion
Dec 27, 2019·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Annelisa M CornelStefan Nierkens
Nov 9, 2019·Investigational New Drugs·Kevin W WellingtonLesetja R Motadi
Jun 22, 2019·BMC Complementary and Alternative Medicine·Luke EsauMandeep Kaur
Oct 22, 2019·European Journal of Immunology·Andrea CossarizzaArturo Zychlinsky
Feb 6, 2021·Bioanalysis·Sonal M ManoharAnusree Nair
Jun 11, 2021·Cellular and Molecular Bioengineering·E Celeste WelchAnubhav Tripathi
Jul 31, 2021·Journal of Proteome Research·Kin-Wing Lui, Sai-Ming Ngai

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Methods Mentioned

BETA
flow cytometry

Software Mentioned

ForeCyt
FlowJo

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