Perforin clone pf 344

The following fluorophore conjugated monoclonal antibodies were used: CD3 (clone SP34-2), CD4 (clone L200), CD95 (clone DX2), Ki67 (clone B56), CD16 (clone 3G8), γδ TCR (clone B1), IFN-© (clone B27), TNFα (clone Mab11) (BD Biosciences), Perforin (clone Pf-344) (Mabtech), CD107a (clone eBioH4A3), CD28 (clone CD28.2); CD8 (clone 3B5) and Granzyme B (clone GB12; Life Technologies). Briefly, lymphocyte single cell suspensions were washed with PBS supplemented with 0.2% heat-inactivated human serum (Sigma), and incubated with different cocktails of fluorophore-labelled monoclonal antibodies during 20 minutes at room temperature [73 (link)], fixed and permeabilized using the FoxP3 permeabilization reagent (eBioscience). After 30 minutes incubation at 4°C, the cells were washed with FoxP3 washing buffer and intracellularly stained with Ki67 and GrzB for 20 minutes. The cells were washed and resuspended in PBS for flow cytometry analysis. For tetramer staining using samples from MamuA*01⁺ macaques, the CM9 tetramer was added to the samples 5 minutes prior to the addition of the antibody cocktail for surface staining [73 (link)]. The samples were acquired on a Fortessa or LSRII flow cytometer (BD Biosciences, San Jose, CA) and the data were analyzed using the FlowJo software platform (Tree Star, Inc., Ashland, OR).

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)].

Surface staining was carried out by standard procedures for our laboratory as described [30 (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), KI-67 (clone EH12.2H7, PE conjugate, custom), CD127 (clone R34.34, PE conjugate, Beckman-Coulter), perforin (clone Pf-344, FITC conjugate, Mabtech, Mariemont, OH). Intracellular staining for perforin and KI-67 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 tetramers to Mamu-A*01 Gag_181–189CM9 and Tat_28–35SL8 (kindly provided by Nancy Wilson and David Watkins, Wisconsin National Primate Research Center, Madison Wisconsin) was performed as described previously [31 (link)]. All acquisitions were made on an LSR II (BD Biosciences) and analyses were done using FlowJo software (Tree Star Inc., Ashland, OR). Isotype-matched controls and/or fluorescence-minus-one (FMO) controls were included in all assays [32 (link)].