AMPK Methods and Protocols

Recovered cells per 1000 beads¼

1000 ðÞnb of events in viable lymphocyte gate %T cell subset

Number of events in bead gate

The percentage of recovery between treated (oligomycin) and

control (medium or DMSO) groups will be further calculated as

[number of cells recovered in oligomycin/number of cells recov-

ered in control]100 (seeFig. 2c for detailed example).

6. Percentage of cell recovery can be plotted in graphs to allow
   visual representation of lymphocyte viability upon metabolic
   stress (seeFig. 3 for the representative experiment depicted
   herein: comparison of graded oligomycin dose effects on B
   cells and helper and cytotoxic T cells from wild-type and con-
   ditional T cell-deficient AMPKα1 mice).

4 Notes

1. It is convenient to use 6μm beads from Polysciences (#07312),
   but any 5–10μm laser-detectable beads would also be suitable.


2. Anti-CD16/32 monoclonal antibody will block binding of
   immunoglobulin to Fc receptors expressed on B cells, mono-
   cytes/macrophages, NK cells, granulocytes, mast cells, and
   dendritic cells.


3. AMPKα1flox/flox-CD4-CRE+mice were generated by breed-
   ing AMPKα1 (Prkaa1)flox/flox mice (kindly provided by
   Dr. Benoit Viollet, Paris, France) with CD4-CRE mice (Jack-
   son Laboratory strain 017336, provided by Dr. Geert Van Loo,
   University of Gent, Belgium), both on a C57BL/6 genetic
   background. The AMPKα1 subunit is the only AMPK catalytic
   isoform present in hematopoietic cell lineages, and disruption
   of the AMPKα1 gene does not lead to a compensatory expres-
   sion of theα2 subunit in the immune system [14].


4. T lymphocytes develop in the thymus where they transiently
   express both CD4 and CD8 co-receptors (double positive
   thymocytes) before giving rise to either CD4+T helper or
   CD8+ cytotoxic T cells. Therefore AMPKα 1 flox/flox-CD4-
   CRE+mice are deficient in AMPK expression in both CD4+
   and CD8+T cell subsets.


5. The bead suspension that we ordered is supplied in the form of
   a dropper bottle. The stock solution corresponds to a concen-
   tration of approximately 200,000 beads/μL. Thoroughly
   shake the vial and take a few drops in a 1.5 mL tube to allow
   easier pipetting. This aliquot solution can be stored at 4Cup
   to 3 months. Check for drying. Once again, thoroughly shake

Flow Cytometry Analysis of Cell Viability upon Metabolic Stress 473