(c) Transfer 250μL of cells from tube 1 to tube 2, mix well,
and transfer again 250μL of twofold diluted cell suspen-
sion from tube 2 to tube 3.
(d) Repeat the 250μL transfer from tube 3 to tube 4 and
from tube 4 to tube 5.
(e) Discard 250μL of diluted cells from tube 5.- Carefully vortex the 6μm polystyrene bead stock solution, and
prepare a 1/250 solution (4μLof6μm beads/mL) in flow
medium (seeNote 5). - Mix well the solution and add 250μL of pre-diluted 6μm bead
stock solution per tube. - Gently mix the tubes and directly analyze by flow cytometry.
- Adjust forward-scattered light (FSC) and side-scattered light
(SSC) to allow simultaneous bead and cell detection: set the
SSC axis into log scale. Beads are very small and you can miss
them in a linear scale (seeNote 6). - Verify correlation between relative bead-based cell count and
theoretical cell dilution curve (see theoretical example in
Fig. 1).
3.2 Oligomycin
Treatment
and Lymphocyte
Subpopulation
Staining
- Prepare oligomycin working solutions from oligomycin stock
solution (seeNote 7):
(a) 20 nM oligomycin: add 2μL of 100μM oligomycin
solution to 10 mL of cell culture medium. Mix well.
(b) 6.6 nM oligomycin: collect 1 mL from the 20 nM oligo-
mycin solution and add 2 mL of cell culture medium.
Mix well.
(c) 2.2 nM oligomycin: collect 1 mL from the 6.6 nM oligo-
mycin solution and add 2 mL of cell culture medium.
Mix well.
(d) DMSO control solution: proceed as for the 20 nM oligo-
mycinsolutionbyadding2μLoftheintermediarysolution
in 10 mL of cell culture medium. Mix well (seeNote 8). - Distribute 250μL of each oligomycin working solution in
cytometer-adapted tubes. Prepare additional tubes containing
250 μL of cell culture medium only. Prepare at least two tubes
for each experimental condition. - Collect spleen cells from control and AMPKα 1 flox/flox-CD4-
CRE+mice as in Subheading3.1 (steps 1– 3 ), and estimate
total spleen cell numbers by trypan blue exclusion. - Adjust spleen cell concentrations to 4 106 /mL, mix well, and
distribute 250μL in tubes containing cell culture medium with
DMSO or oligomycin working solutions. Mix gently. - Incubate 5–6 h in a CO 2 incubator at 37C.
Flow Cytometry Analysis of Cell Viability upon Metabolic Stress 471