Peripheral blood mononuclear cells (PBMCs) were collected in 20 mM EDTA in Eppendorf tubes. PBMCs were spun for 8 minutes at 10,000 rpm at 20°C and serum was removed. RBCs were lysed with ACK Lysis buffer (GIBCO) for 8 minutes at room temperature (rt). If incomplete lysis occurred, cells were spun down for a second time at 12,000 rpm for 30 s and the ACK lysis step was repeated. Mononuclear cells were stained 1:100 with CB101–109/H-2Db-PE tetramer in the presence of Fc block 1:100 (αCD16/32, Tonbo), and monoclonal antibodies at 1:100 specific to CD45 (30-F11, Biolegend), CD8α (53–6.7, ebioscience), LFA-1 (clone H155–78, Biolegend), CD44 (clone IM7, BD), CD62L (MEL-14, Biolegend), PD-1 (J43, Invitrogen), KLRG1 (2F1, Biolegend), Tim-3 (RMT3–23, Biolegend), Lag-3 (C9B7W, Biolegend). CD19 (1D3, BD), F4/80 (T45–2342, BD), and CD11c (N418, BD) were used for a dump channel along with ghost dye BV510 at 1:500 (Tonbo). Antibodies were diluted in FACs buffer (PBS + 2.5% FBS) and cells were stained at room temperature in the dark for 60 minutes and then added to 100 μl of cell counting beads (Sigma Aldrich). Stained cells were fixed with 0.4% paraformaldehyde and stored overnight in the dark at 4°C prior to FACs acquisition using a Fortessa 1770 flow cytometer and Facs Diva software (BD Biosciences) and analyzed using FlowJo software (version 10).
Pd 1 j43
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The PD-1 (J43) is a lab equipment product from Thermo Fisher Scientific. It is a specialized tool used for research purposes. The core function of the PD-1 (J43) is to facilitate the analysis and study of PD-1 (Programmed Cell Death Protein 1) in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cell counting beads, by Merck Group (2 mentions)
αcd16 32, by Cytek Biosciences (2 mentions)
Cd8α 53 6, by Thermo Fisher Scientific (2 mentions)
Lfa 1 clone h155 78, by BioLegend (2 mentions)
Cd44 clone im7, by BD (2 mentions)
Cd62l mel 14, by BioLegend (2 mentions)
Tim 3 rmt3 23, by BioLegend (2 mentions)
Lag 3 c9b7w, by BioLegend (2 mentions)
Cd19 1d3, by BD (2 mentions)
Cd11c n418, by BD (2 mentions)
Fortessa 1770 flow cytometer, by BD (2 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (2 mentions)
Cd45 30 f11, by BioLegend (2 mentions)
Facsdiva software, by BD (2 mentions)
Klrg1 2f1, by BioLegend (2 mentions)
Live dead fixable blue dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Anti cd4 rm4 5, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ xmg1, by BioLegend (1 mentions)
Mhc 2 2g9, by BD (1 mentions)
Cd80 16 10a1, by BD (1 mentions)
11 protocols using pd 1 j43
1
Multiparameter Flow Cytometry of PBMCs
2
Multiparameter Flow Cytometry of PBMCs
3
Phenotyping of Dendritic Cells by FACS
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After homogenizing spleens, cells were treated with erythrocyte lysis buffer, meshed through a 35-μm cell strainer, washed with PBS, and analyzed by FACS.
DCs were phenotyped by using fluorochrome-labeled mAbs directed against CD11c (N418; BioLegend), CD80 (16-10A1; BD Pharmingen, Heidelberg, Germany), CD86 (PO3; Thermo Fisher Scientific), PD-1 (J43, Thermo Fisher Scientific), CTLA-4 (UC10-4B9; BioLegend), CD83 (Michel-19; BioLegend), CD40 (3/23; BioLegend), MHCII (2G9; BD Pharmingen) and PD-L1 (MIH6; AbD Serotec, Puchheim, Germany). Dead cells were excluded using the LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (Thermo Fisher Scientific). Following mAb labeling for 30 min at 4 °C and final washing, cells were analyzed on an LSR II flow cytometer.
To measure intracellular cytokines, cells were stimulated with PMA and ionomycin (1 μg/ml each; Sigma-Aldrich, Taufkirchen, Germany) for 4 h in the presence of 3 μg/ml Brefeldin A (Thermo Fisher Scientific), labeled for CD11c, subjected to fixation and permeabilization and subsequently stained with mAbs against IL-10 (JES5-16E3; BD Pharmingen) and IL-12 (C15.6; BD Pharmingen). IFN-γ in T cells was measured by using fluorochrome-labeled anti-IFN-γ (XMG-1.2, BioLegend) and counterstaining with anti-CD4 (RM4-5, eBioscience, Frankfurt, Germany).
DCs were phenotyped by using fluorochrome-labeled mAbs directed against CD11c (N418; BioLegend), CD80 (16-10A1; BD Pharmingen, Heidelberg, Germany), CD86 (PO3; Thermo Fisher Scientific), PD-1 (J43, Thermo Fisher Scientific), CTLA-4 (UC10-4B9; BioLegend), CD83 (Michel-19; BioLegend), CD40 (3/23; BioLegend), MHCII (2G9; BD Pharmingen) and PD-L1 (MIH6; AbD Serotec, Puchheim, Germany). Dead cells were excluded using the LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (Thermo Fisher Scientific). Following mAb labeling for 30 min at 4 °C and final washing, cells were analyzed on an LSR II flow cytometer.
To measure intracellular cytokines, cells were stimulated with PMA and ionomycin (1 μg/ml each; Sigma-Aldrich, Taufkirchen, Germany) for 4 h in the presence of 3 μg/ml Brefeldin A (Thermo Fisher Scientific), labeled for CD11c, subjected to fixation and permeabilization and subsequently stained with mAbs against IL-10 (JES5-16E3; BD Pharmingen) and IL-12 (C15.6; BD Pharmingen). IFN-γ in T cells was measured by using fluorochrome-labeled anti-IFN-γ (XMG-1.2, BioLegend) and counterstaining with anti-CD4 (RM4-5, eBioscience, Frankfurt, Germany).
4
Ubiquitin Regulation of BECN1 and ZFP91
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Flag-tagged mouse WT BECN1 and hemagglutinin-tagged mouse ZFP91 were cloned into the pcDNA3 vector. His-tagged K48, K63, and K63R mutant ubiquitin were cloned into the pcDNA3 vector. Antibodies for GAPDH, lamin B, p-S6K, p-S6, p-AKT (S473), c-Myc, BECN1, PIK3R4, PIK3C3, and K63 ubiquitin were purchased from Cell Signaling Technology, Inc. HRP-conjugated anti-hemagglutinin antibody (3F10) was from Roche, Inc. Anti-Flag (M2) antibody and anti–β-actin antibody were from Sigma-Aldrich, Inc. Ni–nitrilotriacetic acid agarose was from QIAGEN, Inc. Anti-ZFP91 antibody was from Bethyl Laboratories, Inc. Anti-LC3B antibody was purchased from Abcam, Inc. Anti-p62 antibody was from Proteintech Group, Inc. Antibodies for ubiquitin (P4D1) and NIK (A-12) were from Santa Cruz Biotechnology, Inc. The fluorochrome-conjugated antibodies for CD4 (GK1.5), CD8 (53-6.7), CD44 (IM7), CD62L (MEL-14), Foxp3 (FJK-16s), IFN-γ (XMG1.2), IL-17 (eBio17B7), CD25 (PC61.5), CTLA-4 (UC10-4B9), and PD-1 (J43) were purchased from Thermo Fisher Scientific, Inc. c-Myc (D84C12) rabbit mAb (PE conjugate) and phospho-S6 ribosomal protein (Ser235/236; D57.2.2E) XP Rabbit mAb (allophycocyanin conjugate) were purchased from Cell Signaling Technology, Inc. Rapamycin (R0395), chloroquine diphosphate salt (C6628), leptomycin B solution (L2913), and DCA (D54702) were purchased from Sigma-Aldrich, Inc.
5
T Cell Activation and Phenotyping
6
Multiparametric Flow Cytometry for Immune Cell Analysis
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Unless otherwise stated, cell staining was performed at 4°C. Single-cell suspensions were first incubated with anti-CD16/32 (2.4G2) for 15 min to block antibody Fc receptors. To detect intracellular cytokine production in vitro, cells were stimulated with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) and 500 nM ionomycin for 4 h in the presence of monensin (00-4505-41; eBioscience). Cells were fixed in 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.5% Triton X-100. For transcription factors staining, the Foxp3 Staining Buffer Set (00-5523-00; eBioscience) was used according to the manufacturer’s instruction. Flow cytometry data were collected from the LSR II or FORTESSA (BD Biosciences), and the results were analyzed with the FlowJo 10 software (Tree Star). Antibodies were purchased from several commercial sources indicated below. Antibodies against CD4 (RM4-5), CD25 (PC61.5), CD44 (IM7), CD62L (MEL-14), and PD-1 (J43) were from eBioscience; anti-CXCR5 (2G8), IFN-γ (XMG1.2), T-bet (O4-46), CD138 (281–2), CD19 (1D3), B220 (RA3-6B2), NKG2D (9C11G4), and NKG2A/C/E (20d5) from BD Bioscience; anti-IL-4 (11B11) was from BioLegend; 2.4G2 (PUR001) was from Harlan; and Fixable Viability Dye eFluor 506 was from eBioscience.
7
Multiparameter T Cell Phenotyping
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Cell suspensions were Fc-blocked by using anti-CD16/32 Ab (93; BioLegend). Dead cells were labeled with Live/Dead fixable dead cell stains (Life Technologies). Stainings were performed with the following mAbs: CD3 (17A2; BioLegend), PD-1 (J43; eBiosciences), CD8α (53–6.7; BioLegend), CD62L (Mel-14; eBioscience), Vα2 (B20.1; BioLegend), Vβ5.1/5.2 (MR9-4; BioLegend), and CD25 (PC61, BioLegend). Polyclonal Ab to ORAI1 and STIM1 were purchased from GeneTex. Intracellular stainings were performed by using the PermWash buffer (BD Biosciences). Analyses were performed with an LSR/Fortessa cytometer (BD Biosciences) and analyzed with FlowJo software v.10.1 (Tree Star). For anti–PD-1 treatment experiment, recipient mice were injected at the indicated time with 250 µg blocking anti–PD-1 antibody (RMP1-14; Bio X Cell) or an IgG2Aκ isotype control (2A3, Bio X Cell).
8
Phenotypic Analysis of OT-I CD8+ T Cells
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The following antibodies were used: anti-CD8α (53–6.7; eBioscience/Biolegend/BD), -CD90.1 (OX-7; Biolegend/BD), -CD62L (MEL-14; Biolegend/BD), -CD44 (IM7; eBioscience/Biolegend/BD), -KLRG-1 (2F1; eBioscience), PD-1 (J43; eBioscience), -Ki67 (SolA15; eBioscience), CD127 (A7R34; BD/Biolegend), CD132 (TUGm2; BD), -Bcl-2 (10C4; Biolegend), -IFN-γ (XMG1.2; Biolegend/eBioscience), -TNF-α (MP6-XT22; BD), -CD11c (N418; BD/Biolegend), -CD11b (MI70; BD/Biologend), Gr-1 (RB6-8-C5; Biolegend). Stimulation of CD8+ OT-I T cells with SIINFEKL and subsequent intracellular cytokine staining was performed as described recently [28 (link)]. Samples were measured on FACSCalibur, FACSCanto or LSRII flow cytometers and analyzed by FlowJo software (FlowJo, LLC).
9
Comprehensive Antibody Panel for Immunophenotyping
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Anti–mouse antibodies used in this study included: B220 (RA3-6B2), CD19 (1D3), CD4 (RM4-5), CXCR5 (2G8), and Fas (Jo2) from BD; CD62L (MEL-14), CD11c (N418), Gr-1 (RB6-8C5), Ly5.1 (A20), Ly5.2 (104), CD11b (M1/70), GL7 (GL-7), and PD-1 (J43), from eBioscience; goat anti–mouse IgM−, IgG−, IgG2c− horseradish peroxidase–conjugated, unlabeled, or isotype from SouthernBiotech; CD19 (6D5), CD24 (M1/69), CD23 (B3B4), CD21 (7E9), CD44 (IM7), CD4 (RM4-4), Ly5.2 (104) from BioLegend; PNA (Fl-1071) from Vector Laboratories. Anti–human antibodies used in this study include CD38 (HIT2) and CD27 (L128) from BD; CD19 (HIB19) and IgM (MHM-88) from BioLegend; and IL-6 (MQ2-12A5) from eBioscience.
10
Multicolor Flow Cytometry for Immune Profiling
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