Two-step protocol for preparing adherent cells for high-throughput flow cytometry.
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.
Computational Bioscience Research Center (CBRC), King Abdullah University of Science and Technology (KAUST), Thuwal, Kingdom of Saudi Arabia.
Mandeep
M
Kaur
Computational Bioscience Research Center (CBRC), King Abdullah University of Science and Technology (KAUST), Thuwal, Kingdom of Saudi Arabia.
Luke
L
Esau
D017209:Apoptosis; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D045744:Cell Line, Tumor; D004492:Edetic Acid; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D008297:Male
D005452:Fluoresceins; D005456:Fluorescent Dyes; C029569:diacetyldichlorofluorescein; D004492:Edetic Acid
EDTA; adherent cells; cell detachment; flow cytometry; high-throughput screening