Flow Cytometry Analysis of T Cell Subsets

Following the removal of the olfactory bulb at the indicated time points, tissue was placed in a 2 ml Wheatley Dounce Homogenizer (Fisher Scientific) with 2 ml DMEM media supplemented with high glucose, L-glutamine, and pyruvate (Life Technologies) and 10% FBS. Single cell suspensions were created and processed as previously described [21 (link)]. Briefly, 1/10 the sample homogenate was filtered using a 40-μm nylon mesh filter (Fisher), was pre-incubated with 0.8 μg Fc block (CD16/32) (eBioscience) and then was immunolabeled in 1% FBS/BSA. Total T cells were stained for CD45 eFlour 450 (clone 30-F11), CD3e FITC (clone 145-2C11), CD8a PE (clone 53-6.7), and CD4 APC (clone GK1.5) (all eBioscience). Effector (T-EM) and central memory (T-CM) cells were identified by CD45 eFlour 450, CD3e PE-Cy7, CD4 APC-Cy7, CD8a PE, CD44 APC, and CD62L FITC all from eBioscience as described [21 (link)]. HSV-1-specific T cells were identified by CD3 eFluor 450, CD8a FITC, and either gB-PE, ICP8-A488, or RRI-A488 tetramers (provided by the NIH tetramer facility) as previously described [21 (link)]. All samples were analyzed with the MacsQuant flow cytometer and MacsQuantify software (Miltenyi Biotec).