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

Mandeep Kaur, Luke Esau

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

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.
Original languageEnglish (US)
Pages (from-to)119-126
Number of pages8
JournalBioTechniques
Volume59
Issue number3
DOIs
StatePublished - Apr 3 2015

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