AMPK Methods and Protocols

6. Centrifuge the tubes at 400gfor 5–10 min at 4C.


7. Prepare a “master mix” cell staining solution for lymphocyte
   subpopulation cell staining. Dilute near-IR viability dye
   and fluorescent-coupled anti-mouse TcRβ, CD4, CD8, and
   CD45R monoclonal antibodies, in 50μL of PBS per tube to
   stain (see Note 9). Total volume solution to be
   prepared¼[number of tubes to stainþ1] 50 μL(seeNote
   10 ).


8. Discard the cell supernatant (seeNote 11) and gently resus-
   pend the cells in 50μL of master mix cell staining solution.


9. Incubate at 4C for 25 min.


10. Add 2 mL of cold PBS and centrifuge the tubes at 400gfor
    5–10 min at 4C.


11. Carefully homogenize the 6μm polystyrene bead solution, and
    prepare a 1/250 solution (4μLof6μm beads/mL) in flow
    medium (seeNote 5).
    Total volume solution to be prepared¼[number of tubes to
    stainþ1] 500 μL.


12. Discard the cell supernatant (seeNote 11), and gently resus-
    pend the cells in 500μLof6μm polystyrene bead-containing
    flow medium (seeNotes 12and 13 ).


13. Adjust forward-scattered light (FSC) and side-scattered light
    (SSC) to allow simultaneous bead and cell detection: regulate
    SSC axis into log scale (seeNote 6). Gently vortex the tubes
    before analyzing by the cytometer.

3.3 Flow Cytometry
Analysis
and Lymphocyte
Subset Viability
Determination

A representative example and calculation procedure are shown in

Fig. 2.

1. From the FSC/SSC dot plot diagram, draw the bead and
   lymphocyte gates.


2. Select “lymphocyte gate” and open a FSC-A/FSC-H dot plot
   diagram, and then draw a single-cell gate (seeNote 14).


3. Select “single-cell gate” and open the FSC/viability marker dot
   plot diagram. Draw a new gate¼single live lymphocytes.


4. From this new gate, open the fluorescent marker-associated
   diagrams (i.e., in Fig. 2a, b:TcRβ versus CD45R). Draw
   gates corresponding to T lymphocytes (TcRβ+) and B lympho-
   cytes (CD45R+).


5. From the different gate/plot previously described insteps 1– 4 ,
   record the numbers of beads, numbers of viable lymphocytes,
   and percentage of T cell subset. The data collected in the Fig. 2
   will allow calculation of the relative recovery (i.e., for 1000
   beads) of T lymphocyte subset, in each experimental condition,
   according to the formula:

472 Se ́bastien Denanglaire et al.