the 1.5 mL tube by pipetting up and down before taking a few
microliters.- Be careful of the instrument threshold settings. Beads are
indeed very small and you can miss them if your threshold is
set too high. - The final volumes of each solution should be adapted depend-
ing on the number of experimental condition to be tested.
Perform each experimental condition at least in duplicate. - Do not exceed a final concentration of 0.1% (v/v) DMSO in
any of the samples, to limit interference by this solvent on the
biological response analyzed. - Dilution of fluorescent-labeled antibodies and viability dye in
this particular example:
Near-IR viability dye, 1/1000 (Life Technologies, L10119);
anti-mouse CD16/32, 1/200 (BioXCell, CUS-HB-197);
anti- TcRβ-PerCP-Cy5.5, 1/100 (BD, 560657); anti-CD4-
PECy7, 1/250 (BD, 552775); anti-CD8-PE, 1/250 (BD,
553033); anti-CD45R-FITC, 1/100 (BD, 553087). - Additional tubes with control (untreated) cells stained by sin-
gle antibody only are required to allow proper instrument
calibration (for details, seehttp://flowjo.typepad.com/the_
daily_dongle/2017/03/demystifying-compensation-in-
flowjo.html or http://www.drmr.com/compensation/inde
xDetail.html). - This step is very crucial: it is important to discard supernatant
with a minimal loss of cells and to proceed in exactly the same
way for all the tubes for adequate comparison. - Homogenate the bead solution immediately before distribu-
tion as beads will rapidly sediment to the bottom of the tube. It
is important to distribute the same amount of beads in each
experimental tube to ensure reliability and reproducibility of
the results. - If the identification of lymphocyte subsets requires extensive
centrifugation steps and treatments that may lead to loss of
viable cells during the procedure (i.e., cell fixation/permeabi-
lization for intracellular transcription factor staining), it is
recommended to separate the cell counting procedure and
the estimation of cell subset relative frequencies. After meta-
bolic stress treatment, cells may therefore be divided in two
tubes:
(a) In tube 1, cells are mixed with viability dye and 6μm
polystyrene beads (for total viable cell counting).
(b) Tube 2 is used for classical flow cytometry analysis (esti-
mation of subset cell frequencies).
474 Se ́bastien Denanglaire et al.