Surface markers and intracellular Ki67were stained for phenotyping and measuring in vivo adoptively transferred and proliferating anti-SHIV T Cells. After PBMCs were isolated from the blood and tissue samples, cells were washed twice with PBS and stained with LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Thermo Fisher SCIENTIFIC) for 20 min at room temperature followed by staining with the following antibodies: CD3-Cy7-APC (Clone: SP34.2, BD Biosciences), CD4-BV605 (Clone: OKT4, BioLegend), CD8-BV711 (Clone: RPA-T8, BioLegend), CD28-Cy5PE (Clone: 28.2, BD Biosciences), CD45RA-Cy7-PE (Clone: L48, BD Pharmingen), CD69-ECD (Clone: TP1.55.3, Beckman Coulter), CCR7-Alexa Flour 680 (Clone: 150503, BD Pharmingen) and HLA-DR-BV421 (Clone: LN3, Biolegend). After surface staining, cells were permeabilized with Foxp3 / Transcription Factor Staining Buffer Set (eBiosciences) for 20 min at room temperature, then intracellular stained with anti-Ki67-PE (Clone: B56, BD Pharmingen).
Hla dr bv421
Manufactured by BioLegend
Sourced in United States
HLA-DR-BV421 is a fluorochrome-conjugated antibody that binds to the HLA-DR antigen, a major histocompatibility complex (MHC) class II cell surface receptor. It is used in flow cytometry applications to identify and quantify HLA-DR-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Cd69 ecd, by Beckman Coulter (1 mentions)
Cd8 bv711, by BioLegend (1 mentions)
Cd3 cy 7apc, by BD (1 mentions)
Cd28 cy 5pe, by BD (1 mentions)
Anti ki67 pe, by BD (1 mentions)
Cd45ra cy7pe, by BD (1 mentions)
Cd4 bv605, by BioLegend (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Cd81 pe cy7, by BioLegend (1 mentions)
Cd9 pe, by BioLegend (1 mentions)
Anti cd16 cd32, by BioLegend (1 mentions)
Pd l1 bv650, by BioLegend (1 mentions)
7 aad viability dye, by BioLegend (1 mentions)
Fixable viability stain 700, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Cd56 bv650, by BioLegend (1 mentions)
Cd11c pe dazzle594, by BioLegend (1 mentions)
Cd80 bv711, by BioLegend (1 mentions)
Cd86 buv737, by BD (1 mentions)
7 protocols using hla dr bv421
1
Phenotyping and Proliferation of Anti-SHIV T Cells
2
Imaging Flow Cytometry Characterization of EVs
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Imaging flow cytometry (IFCM) characterisation of EVs was adapted from a previously published protocol27 (link). EVs were analysed using an Amnis ImageStreamX Mark II Flow Cytometer with the following antibodies, all from Biolegend: anti-CD63-FITC (CD63-FITC; 1:150, Cat.#:353006); CD9-PE (1:1500, Cat.#:312106); CD81-PE-Cy7 (1:150, Cat.#:349512); HLA-DR-BV421 (1:600, Cat.#:307636) and ADAM10-APC (1:150, Cat.#:352706)17 (link),26 (link),27 (link). EV-free IFCM buffer, unstained EVs, single-stained controls and FMO (fluorescence-minus-one) controls were run in parallel. Data analysis used IDEAS v6.2 (Amnis/Luminex, Seattle). EVs were gated as SSC-low vs fluorescence, then as non-detectable brightfield (fluorescence vs Raw Max Pixel Brightfield channel) (Supplementary Fig. S5 ). Gated EVs were confirmed in the IDEAS Image Gallery.
3
Comprehensive Immune Cell Profiling of HPSCC
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Fresh tumor tissues and the corresponding adjacent tissues were harvested from patients with HPSCC and washed in PBS to remove the blood. Next, the tissues were cut into 2-mm pieces and digested using digestive enzymes [10% collagenase/hyaluronidase + 90% (DMEM + 5% FBS) + 1 mg/mL DNase I] at 37 °C on a shaking table (140 rpm) for 30 min, centrifuged at 300 × g at 4 °C for 5 min, and filtered with a 70-μm strainer to prepare a single-cell suspension.
Single-cell suspensions were resuspended in FACS buffer (PBS + 2% FBS) and incubated with anti-CD16/CD32 (BioLegend, 156603) for 20 min on ice. For cell membrane staining, the samples were first stained with Dead Cell Stain (Thermo Fisher, USA) for 15 min at 25 ℃ and then incubated with the cell surface markers CD4 (FITC), CD127 (APC), CD11b (PerCP/Cy5.5) from eBioscience, CD45 (Spark BlurTM550), CD3 (AF700), CD25 (PE/Cy7), PD-1 (PE/Dazzle594), CCR7 (Spark NIRTM685), CD11C (BV510), HLA-DR (BV421), and PD-L1(BV650) from Biolegend, and LAMP3 (PE, BD) for 30 min at 4 °C. Spectroscopic flow cytometry was performed for detection and analysis.
Single-cell suspensions were resuspended in FACS buffer (PBS + 2% FBS) and incubated with anti-CD16/CD32 (BioLegend, 156603) for 20 min on ice. For cell membrane staining, the samples were first stained with Dead Cell Stain (Thermo Fisher, USA) for 15 min at 25 ℃ and then incubated with the cell surface markers CD4 (FITC), CD127 (APC), CD11b (PerCP/Cy5.5) from eBioscience, CD45 (Spark BlurTM550), CD3 (AF700), CD25 (PE/Cy7), PD-1 (PE/Dazzle594), CCR7 (Spark NIRTM685), CD11C (BV510), HLA-DR (BV421), and PD-L1(BV650) from Biolegend, and LAMP3 (PE, BD) for 30 min at 4 °C. Spectroscopic flow cytometry was performed for detection and analysis.
4
Multicolor Flow Cytometry of MSCs
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Flow cytometry was performed with LSRFortessa (Becton-Dickinson, Heidelberg, Germany). Cells were stained with 7-AAD viability dye (BioLegend, San Diego, CA, United States) and the following antibodies (all from BD Biosciences unless otherwise noted): anti–CD45-V500 (HI30), anti–CD34-PE (581), anti–CD14-FITC (M5E2, BioLegend), anti–CD19-APC (SJ25C1), anti–HLA-DR-BV421 (G46-6), anti–HLA-ABC-APC (G46-2.6), anti–CD29-BV510 (MAR4), anti–CD59-FITC (p282(H19), BioLegend), anti–CD105-PE-CF594 (266), anti–CD140b-PE (28D4), anti–CD44-AF700 (G44-26), anti–CD90-PE-Cy7 (5E10), and anti–CD73-BV605 (AD2), these comprising two separate tubes of multicolor panel (MCP) I, analyzing the MSCs with regard to the ISCT MSC criteria (Dominici et al., 2006 (link)). To identify subpopulations, MCP II was developed and established, herein staining cells with Fixable Viability Stain 700 (BD Biosciences) and the following antibodies (all from BD Biosciences unless otherwise noted): anti–CD140a-BV605 (αR1), anti–CD271-BV421 (C40-1457), anti–CD146-BV510 (P1H12), anti–CD119-PE (GIR-208), anti–CD106-PE-Cy5 (51-10C9), and anti–MSCA-1-APC (W8B2, Miltenyi Biotec). The MSCs were analyzed at P3. For multicolor flow cytometry gating strategy (see Figure 1 ).
5
Flow cytometry analysis of PBMCs
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Peripheral blood mononuclear cells (PBMCs) isolated from buffy coats (Red Cross) using standard gradient density centrifugation were spinoculated with vectors at 2671 g for 2 hours, RT, MOI5. After 20 hours, PBMCs were harvested using 0.05% Trypsin-EDTA, seeded in 96-well plates, washed using FACS buffer (PBS (Phosphate Buffered Saline), 2.5% FBS, 10 mM EDTA) with 0.01% sodium azide, stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher) and incubated for 1 hour in CD56-BV650, CD19-BV650, HLA-DR-BV421, CD14-AF700, CD11c-PE-Dazzle594, CD303-PerCP-Cy5.5, CD1c-APC-Cy7, CD141-PE-Cy7, CD80-BV711 and CD40-BV605 (BioLegend), CD86-BUV737, CD16-BV786 (BD Biosciences). After washing, cells were fixed with Cytofix (BD Biosciences), resuspended in FACS buffer, and analyzed using a BD Fortessa flow cytometer and FlowJo v10.8 Software (BD Life Sciences).
6
Flow Cytometry Analysis of PBMCs
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PBMCs from a subset of patients undergoing THA or TKA were analyzed by flow cytometry (n = 19). Within 60 min after each draw, blood was layered over Ficoll-Paque PLUS, leukocytes were collected from the interface, and remaining red blood cells (RBCs) were lysed using RBC Lysis Buffer (BioLegend; San Diego, CA, USA). After lysis, cells were washed, incubated with Human FcR Binding Inhibitor (eBioscience; San Diego, CA, USA), and stained with anti-human CD8a-AlexaFluor488, CD4-PE, CD66b-PE-Dazzle, CD25-APC, CD45-APC-Cy7, CD127-PerCP-Cy5.5, CD14-PE-Cy7, HLA-DR-BV421, CD15-BV510, CD19-BV605, CD33-BV711, and CD16-BV650 (all from BioLegend; San Diego, CA, USA). Dead cells were excluded using a LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Life Technologies; Eugene, OR, USA) and analysis was performed using BD FACS-DIVA software, as previously described with the gating strategy depicted in Supplementary Figure S1 [16 (link)].
7
Isolation and Characterization of Distinct CD4+ T Cell Subsets
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To obtain naive, CM/HLA-DR±, TM/ HLA-DR±, EM/HLA-DR± cells, peripheral blood mononuclear cells were obtained by Lymphoprep (STEMCELL Technologies) density gradient centrifugation. Total CD4+ T cells were isolated by magnetic negative selection (STEMCELL Technologies). Cells were then sorted using a FACSAria (BD Biosciences) using the following antibodies: CD3-FITC (clone UCHT1, BioLegend), CD4-BV650 (clone RPA-T4, BioLegend), CD45RA-PE (clone HI100, BioLegend), CCR7-PECy7 (clone G043H7, BioLegend), CD27-APC (clone M-T271, BioLegend), HLA-DR-BV421 (clone L243, BioLegend), and Live/Dead Aqua Marker (Fisher Bioscience) (Supplementary Figure 1C ).
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