Caltag fix perm

Surface staining was carried out by standard procedures for our laboratory as described [56 (link)]. Except where noted, all reagents were obtained from BD Biosciences (San Diego, CA) and included monoclonal antibodies to the following molecules: CD3 (clone SP34-2, APC-Cy7 conjugate) CD4 (clone SK3, PerCP-Cy5.5 conjugate), CD8α (clone RPA-T8, Alexa700 conjugate) CD28 (clone CD28.2, PE-Texas Red conjugate, Beckman-Coulter, Fullerton, CA), CCR7 (clone 150503, Pacific Blue conjugate, custom) CXCR3 (clone 1C6, PE-Cy5 conjugate), Ki-67 (Clone B56, PE conjugate), CD127 (clone R34.34, PE conjugate, Beckman-Coulter), perforin (clone Pf-344, FITC conjugate, Mabtech, Mariemont, OH). Intracellular staining for perforin expression was performed using Caltag Fix & Perm (Invitrogen, Camarillo, CA) according to the manufacturer’s suggested protocol. Enumeration of SIV-specific cells using PE- or APC-conjugated pentamers to Mamu-A*01 Gag_181-189CM9 and Tat_28-35SL8 (Proimmune, Oxford, UK) was performed as described previously [57 (link)]. All samples were analyzed using an LSR II (BD Biosciences), and analyses were performed using FlowJo software (Tree Star Inc., Ashland, OR). Isotype-matched controls and/or fluorescence-minus-one (FMO) controls were included in all assays [58 (link)].

The LIVE/DEAD Fixable Aqua Dead Cell Stain Kit was applied to excluding the dead cells. Cell surface staining was conducted using the following monoclonal antibodies including CD45 (BV605 conjugate, clone D058-1283, BD Pharmingen), CD3 (Alexa 700 conjugate, clone SP34.2, BD Pharmingen), CD4 (BB700 conjugate, clone L200, BD Pharmingen), CD8 (Pacific Blue conjugate, clone RPA-T8, BD Pharmingen), CD95 (BV711 conjugate, clone DX2, BD Pharmingen), CD28 (PE-Cy7 conjugate, clone 28.2, ThermoFisher), CD69 (ECD conjugate, clone TP1.55.3, Beckman Coulter), and CD195 (BV650 conjugate, clone 3A9, BD Pharmingen). For intracellular staining, cells were permeabilized using Caltag Fix & Perm (Invitrogen), then stained with monoclonal antibodies including Ki67 (PE conjugate, clone B56, BD Pharmingen) and caspase 3 (BUV395 conjugate, clone C92-605, BD Pharmingen). Fluorescent labeled inhibitor probe FAM-YVAD-FMK (ImmunoChemistry Technologies) which could covalently bind to the activated caspase 1, was applied to detecting the cells containing activate caspase 1. Autophagy probe (ImmunoChemistry Technologies) which fluoresces red when inserting into the lipid membrane of autophagosomes and autolysosomes was applied in the study. After staining, cells were washed and fixed by 2% paraformaldehyde. All data were acquired on BD LSRII flow cytometer and analyzed by FlowJo software (version 9.9.5).