further explain the cytometry analysis procedure and viable cell
calculation for T lymphocytes identified by flow cytometry
(Fig.2). Finally, with this technique, we illustrate the selective
sensitivity of AMPK-deficient CD4+and CD8+T lymphocytes to
a mitochondrial metabolic stress (Fig.3).
Importantly, this technique is also applicable to the assay of any
cell type viability in response to panels of drugs.2 Materials
2.1 Solutions,
Buffers, and Media
- Red blood cell lysis solution: 17 mM Tris–HCl, pH 7.65,
144 mM NH 4 Cl. Mix 9 volumes of 0.16 M NH 4 Cl and
1 volume of 0.17 M Tris–HCl, pH 7.65. - Cell culture medium: RPMI 1640 supplemented with 5% (v/v)
fetal bovine serum, 2 mML-glutamine, 1 mM Na-pyruvate,
100 μM nonessential amino acids, and 50 U/mL penicillin/
streptomycin.
Fig. 1Correlation of flow cytometry bead-based cell counting and theoretical cell dilution curve. Serial twofold
dilutions of splenocytes were mixed with equal number of 6μm beads. (a) Cytometer profiles showing 6μm
beads and viable cells. Acquisition was stopped at 500 events in the bead gate. Number of acquired
lymphocytes is indicated in each cell gate (mean duplicatesSD). (b) Experimental cell counts from
(a) were plotted on theoretical curve of twofold dilutions
Flow Cytometry Analysis of Cell Viability upon Metabolic Stress 467