Pe conjugated anti cd3 clone sp34

All CD11b⁺ myeloid cells were labeled with PE-conjugated anti-CD3 clone SP34 (BD Bioscience) and fluorescein isothiocyanate-conjugated anti-CD11b clone Bear1 (Beckman Coulter, Brea, CA) to assess the selection efficiency. Purified CD4⁺ T cells were stained with antibodies for HLA-DR clone L243 (BioLegend), CD3 clone SP34-2 (BD Bioscience), CD4 clone OKT4 (BioLegend), CD8 clone RPA-T8 (BioLegend), and TCRγδ clone B1.1 (eBioscience). Cells were stained for 20 min at room temperature in 100 μl phosphate-buffered saline–2% FBS and fixed for 10 min with Fix/Lyse buffer (Becton Dickinson, Franklin Lakes, NJ). After fixation, samples were analyzed in a BD LSRFortessa flow cytometer using DIVA software (Becton Dickinson, Franklin Lakes, NJ). The gating of CD3⁺ T cells was easily visualized as small CD3⁺ nonautofluorescent cells. All data were analyzed using FlowJo software. CD4⁺ T cell and monocyte counts were analyzed as previously described (38 (link)).

TCRβ RNA was quantitated by qRT-PCR using a QuantiTect kit (Qiagen), the above-described primers, and the probe 5′-/56-FAM/ACT TCC GCT/ZEN/GCC AAG TCC AGT TCT AT/3IABkFQ/-3′ (where 56-FAM is 6-carboxyfluorescein, 3IABkFQ is 3′ Iowa Black FQ, and Zen is an internal quencher [Integrated DNA Technologies, Coralville, Iowa]), based on sequence analyses of macaque TCRβ RNA. Cycling conditions were as follows: 50°C for 30 min, 95°C for 15 min, and 45 cycles of 94°C for 15 s, 55°C for 15 s, and 60°C for 30 s. 18S rRNA was multiplexed with the TCRβ RNA to control for cell counts. To determine the average number of TCRβ copies per CD3⁺ T cell, PBMCs from seven uninfected macaques were labeled with phycoerythrin (PE)-conjugated anti-CD3 clone SP34 (BD Bioscience, San Jose, CA) and magnetically separated using an EasySep PE positive selection kit (Stemcell Technologies, Vancouver, BC, Canada). The purity of the cells was confirmed by flow cytometry (see Fig. 2A). A minimum of 4 aliquots of a million CD3⁺ T cells from each macaque was used to isolate RNA that was analyzed by qRT-PCR for TCRβ RNA, and the number of copies of TCRβ RNA per macaque CD3⁺ T cell was calculated (see Fig. 2B).