where infiltrating tumor-specific lymphocytes may face severe hyp-
oxia and/or reduced glucose availability [10–13].
Studies on lymphocytes deficient for AMPK have revealed that
this enzyme plays an important role in promoting lymphocyte
resistance to an energy crisis [14]. In particular, AMPK-deficient
lymphocytes are unable to maintain adequate ATP levels and cell
viability during metabolic mitochondrial stresses induced by ATP
synthase inhibition [14].
Recent evidence indicates that T lymphocyte subsets display
distinct metabolic profiles with effector T helper (Th) cells expres-
sing strong glycolytic metabolic profile, whereas regulatory T cells
(Tregs) display increased mitochondrial fatty acid oxidation-based
metabolism [15, 16]. Thus local metabolic stresses may differently
affect distinct lymphocyte subsets.1.1 Lymphocyte
Identification by Flow
Cytometry
Lymphocyte cell types differ by the expression of selective surface
molecules (e.g., CD for cluster of differentiation) [17]. Panels of
fluorescent anti-CD-specific antibodies represent a powerful tool
for the identification of lymphocyte subpopulations by flow cyto-
metry and offer interesting potential for measuring selective viable
cell subset recovery in mixed populations exposed to metabolic
stress.1.2 Lymphocytes
Counting
Manual counting of recovered cell (i.e., trypan blue exclusion)
combined with cell subset distribution information (given by flow
cytometry) is often used to estimate cell recovery of a particular lym-
phocyte population without extensive cell subset purification proce-
dures(numbersofviablecellsfrom“subsetA”¼totalnumberofviable
cells x percentage of cells in “subset A”). However, manual counting is
time consuming and difficult to apply to a large-scale analysis, in
particular in the setting of kinetic studies, and most standard cyt-
ometers are not equipped with an absolute cell counting mode.1.3 Protocol Outline Here we describe a method allowing simultaneous lymphocyte
subset identification and relative cell counting by flow cytometry.
Subheading3.1 (illustrated in Fig.1) is devoted to the validation of
the technique in the local setup. The method relies on adding a
fixed amount of easily recognizable (due to distinct size and light
opacity) polystyrene beads to cell suspensions resulting from a serial
twofold dilution of splenocytes. Plotting the relative cell numbers
corresponding to the same number of beads and theoretical cell
dilution curve reveals a very good correlation over a wide range of
cell numbers (Fig.1b). In Subheading3.2, we will describe the
step-by-step procedure allowing the analysis of T helper lympho-
cyte (CD4+), cytotoxic T lymphocyte (CD8+), and B lymphocyte
(CD45R+) viability in unfractionated spleen cells from wild-type
and T cell conditional AMPKα1-KO mice treated with the F0/F1
ATP synthase inhibitor oligomycin. In Subheading3.3, we will
466 Se ́bastien Denanglaire et al.