For
in vitro proliferation experiment, gBT-1 CD8+ T cells from adult and neonatal mice were isolated by positive magnetic selection. Purified cells were labeled with CFSE dye
36 (link), resuspended in
RPMI 1640 (Lonza, Basel, Switzerland) supplemented with 10%
heat inactivated serum (PAA Laboratories, Pasching, Austria) and stimulated with peptide (10
−9M) in the presence of IL-2 (100U/ml). Cells were collected at 4, 16, 20, 40, 52, and 64 hours post-stimulation. To determine total cell number, 1×10
4 unlabeled calibrite beads (BD Biosciences, Mountain View, CA) were added to samples prior to staining procedures. Cells were stained with
monoclonal antibodies to anti-CD8α (53–6.7, cat # 48–0081–82, eBiosciences, San Diego, CA),
anti-CD4 (GK1.5, cat # 56–0041–82, eBiosciences), anti
-CD62L (MEL-14, cat # 562404, BD Biosciences, San Jose, CA) and
anti-Ly6c (HK1.4, cat # 47–5932–82, eBiosciences). To determine cell viability, cells were stained with
fixable viability dye-e780 (65–0856–14, eBiosciences) according to manufacturer’s instruction. The number and phenotype of cells in each division was estimated using FlowJo’s proliferation analysis (Treestar, OR). We used four replicates of adult and four neonatal cells in our
in vitro experiment (based on our pilot study).
Reynaldi A., Smith N.L., Schlub T.E., Venturi V., Rudd B.D, & Davenport M.P. (2016). Modeling the dynamics of neonatal CD8+ T cell responses. Immunology and cell biology, 94(9), 838-848.
