CD4 T‐cell purity, composition and transfection efficiency were assessed by flow cytometry (BD LSR II). Purity and composition panel (stained in 250 μl): CD4 APC (#357408), CCR4 BV421 (#359414), CCR6 AF700 (#353434), CD3 FITC (#300306), CD62L PerCP/Cy5.5 (#304824), CXCR3 PE/Dazzle 594 (#353736) and CD45RA PE/Cy7 (#304126)—all used at 1:100 dilution (all BioLegend), Zombie Aqua Fixable Viability Kit (BioLegend) and Fc receptor blocking reagent (Miltenyi). Transfection efficiency panel: EGFP, CD4 APC (as above), Zombie Aqua Fixable Viability Kit, Fc receptor blocking reagent. Data were gated using FlowJo v10 (BD).
Cd62l percp cy5
Manufactured by BioLegend
Sourced in United States
CD62L-PerCP/Cy5.5 is a fluorochrome-conjugated antibody that binds to the CD62L (L-selectin) cell surface marker. CD62L is a cell adhesion molecule involved in the homing of lymphocytes to peripheral lymph nodes.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 fitc, by BioLegend (3 mentions)
Cd4 apc, by BioLegend (2 mentions)
Ccr4 bv421, by BioLegend (1 mentions)
Cd3 fitc, by BioLegend (1 mentions)
Cd45ra pe cy7, by BioLegend (1 mentions)
Zombie aqua fixable viability kit, by BioLegend (1 mentions)
Lsr 2, by BD (1 mentions)
Fc receptor blocking reagent, by Miltenyi Biotec (1 mentions)
Cd45 bv421, by BioLegend (1 mentions)
Cd8 alexa fluor 700, by BioLegend (1 mentions)
Nk1.1 bv711, by BioLegend (1 mentions)
Ifn γ bv510, by BioLegend (1 mentions)
Il 4 bv605, by BioLegend (1 mentions)
Il 17 percp cy5, by BioLegend (1 mentions)
Cd69 percpcy5, by BioLegend (1 mentions)
Live dead staining kit, by Thermo Fisher Scientific (1 mentions)
Foxp3 fitc, by BioLegend (1 mentions)
Cd3 pe cy7, by BioLegend (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Diva software, by BD (1 mentions)
8 protocols using cd62l percp cy5
1
Comprehensive CD4+ T-cell Characterization
2
Intracellular Staining and Cytokine Detection
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For intracellular staining of phosphor-proteins, cells were fixed with 2% paraformaldehyde, followed by permeabilization in 95% methanol. The following antibodies were used: anti-p-PDHE1α (AP1062; Calbiochem, Darmstadt, Germany) and a fluorescein isothiocyanate conjugated antirabbit secondary antibody (Santa Cruz Biotechnology, Dallas, TX). For staining of intracellular cytokines/transcription factors, either the Mouse Foxp3 Buffer Set 560409 (BD Biosciences, Franklin Lakes, NJ) or the Foxp3/Transcription Factor Staining Buffer Set 00-5523-00 (eBioscience, San Diego, CA) was used along with the following antibodies: CD45-BV421, CD3-PECy7, CD4-APC, CD8-Alexafluor700, NK1.1-BV711, IFN-γ-BV510, IL-4-BV605, IL-17-PerCP-Cy5.5, CD25-BV785, Foxp3-FITC, CD69-PerCPCy5.5, and CD62L-PerCPCy5.5 (all from BioLegend, San Diego, CA). Cell viability was measured using a Live/Dead staining kit (Thermo Fisher). Cell death was measured using an FITC/Annexin V Apoptosis Detection Kit (556547; BD Biosciences).
3
Comprehensive Tumor Immune Profiling
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Tumor cells isolated from mice were thawed and stained with LIVE/DEAD Fixable Violet Dead Staining Kit (ThermoFisher). Subsequently, the cells were divided and stained with cocktails of fluorochrome-conjugated monoclonal antibodies: CD3 PE-CF594, CD19 PE-CF594, CD49b PE-CF594 (all from BD Biosciences), CD45 BV605, CD11b PerCP-Cy5.5, CD11c BV650, F4/80 AlexaFluor 700, Ly6C PE, Ly6G APC-Cy7, MHC II FITC, CD80 PE-Cy7 (all from Biolegend) for myeloid cell identification and CD45 BV605, CD3 BV650, CD4 FITC, CD8 APC-Fire, CD25 PE, CD44 PE-Cy7, CD62L PerCP-Cy5.5 (all from BioLegend) for lymphocytes identification. Then, the cells were fixed using FoxP3 Fixation Permeabilization Staining Kit (eBioscience). Tumor cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or FoxP3 APC (eBioscience) antibodies, respectively. The analysis was performed using FACSFortessa flow cytometer with Diva software (Becton Dickinson).
4
Multiparameter Flow Cytometric Analysis of Murine Splenocytes
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Spleens were disaggregated into single cell suspensions by using a plunger from a sterile syringe to mince and stained for flow cytometric analysis. Samples were blocked with anti‐CD16/32 (Biolegend) for 30 min at 4°C and the following anti‐mouse primary antibodies incubated for 1 h at 4°C. All antibodies were from Biolegend: CD3:Pacific Blue, CD4:FITC, CD44:AF700, CD62L.PerCP/Cy5.5, PD1:PE, CD153:APC, PE isotype Ctrl, PerCP/Cy5.5 isotype Ctrl, APC isotype Ctrl (Figure. 2d ). Single‐stained samples were prepared as compensations. Cells were washed twice and resuspended in PBS/2% FCS for analysis on LSR Fortessa using FlowJo v10.8 software (BD Life Sciences). Apoptosis induction by ABT‐263 was analyzed by flow cytometer using Annexin V‐FITC/PI staining kit (Biolegend).
5
Flow Cytometry Analysis of Mouse Immune Cells
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Single cell suspensions prepared from spleen and liver were analyzed using flow cytometry (FACS) (28 (link)). The antibodies used for FACS staining including anti-mouse CD3-PE or CD3-FITC, CD4-PerCP/Cy5.5 or CD4-FITC, CD8-APC or CD8-FITC, CD69-FITC, CD62L-PerCP/Cy5.5, CD44-PE, CD19-APC, NK1.1-PE or NK1.1-PErCP/Cy5.5, Ly6C/Ly6G-Gr1, F4/80-PerCP/Cy5.5, IFNγ-PE, and TNFα-APC were purchased from Biolegend (San Diego, CA, USA). Anti-mouse CD16/32 FcR blocking antibody was also from Biolegend. For intracellular cytokine staining, cells were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Beyotime, Haimen, China) and 500 ng/ml ionomycin (Beyotime) for 4 h in the presence of 1× brefeldin A (Biolegend). Cells were harvested and stained with surface antibodies, then were washed, fixed with 4% paraformaldehyde, permeabilized with 1% saponin and stained with intracellular cytokine antibodies. FACS was performed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using Flowjo software (Tree Star, Ashland, OR, USA).
6
Comprehensive Phenotypic Analysis of Immune Cell Subsets
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For phenotypic analysis, cells were surface-stained with: TIGIT-BV421, CD103-BV605, CD69-BV711, PD-1-BV786, CD28-FITC, CD49d-PE, CD122-PE-Cy7, DNAM-1 (CD226)-APC, CX3CR1-Alexa700, CD27-APC-Cy7, CTLA-4 (CD152)-BV421, TIM3-BV605, OX40-BV711, CD8-BV786, 2B4 (CD244)-FITC, CD62L-PerCP-Cy5.5, ICOS-PE-Dazzle, CD43-PE-Cy7, CD44-Alexa700, CD25-APC-Cy7 (BioLegend), CD8-PacOrange, CD127-APC (ThermoFisher), CD4-BUV395, CXCR3-BV510 (BD). For Fc staining, cells were incubated with biotinylated CD16/CD32 (2.4G2, BD Biosciences), washed twice, and stained with streptavidin-PerCP-Cy5.5 (BioLegend). For transcription factor staining, cells were permeabilized using a FoxP3/transcription factor kit (Invitrogen) and stained with FoxP3-PE (ThermoFisher). Absolute numbers were calculated using CountBright Absolute Counting Beads according to the manufacturer’s instructions (ThermoFisher). Samples were analyzed on an LSRFortessa flow cytometer (BD). Data were analyzed using FlowJo software (Tree Star).
7
Immunophenotyping of Chronic Lymphocytic Leukemia
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PBMCs were separated by density centrifugation of fresh CLL blood samples. PBMCs were incubated with directly conjugated mAbs for 20 minutes at room temperature. Flow cytometric analysis was performed after erythrolysis with IOTest® 3 Lysing Solution (Beckman Coulter, IM3514) and washing steps. Monoclonal antibodies for analysis were provided by Biolegend: anti-human PD-1-Brilliant Violet 421 (329920),CXCR3-APC (353708), CXCR5-PerCP-Cy5.5 (356910), CCR4-PE-Cy7 (359410), CD62L-PerCP/Cy5.5 (304824) and CD226-PerCP/Cy5.5 (338314). Monoclonal antibodies provided by ThermoFisher included TIGIT-PE (12–9500-42), CD4-PE-Cyanine7 (25–0048-42), CD4-eFluor450 (48-0048-42), CD4-APC (MHCD0405), 2B4-APC (17-5837-42), CD3-Alexa Fluor 700 (56-0032-82), CD3-FITC (11-0039-42), CD45RA-APC-eFluor780 (47-0458-42), CD25-PE-Cyanine7 (25-0259-42), CD127-APC-eFluor780 (47-1271-82), CD155-PE (12-1550-41), CD112-APC (17-1128-42), CD19-FITC (11-0199-42), rhAnnexin V-FITC (BMS147FI), 7-AAD Viability Staining Solution (00-6993-50), TIM-3-PerCP-eFluor 710 (46-3109-42) and CD8-Pacific Orange (MCD0830). For viability assays, rhAnnexin V/FITC, 7-AAD Viability Staining Solution was used. Data acquisition was performed on Gallios™ Flow Cytometer research system (Beckman Coulter) and data analysis was performed using Kaluza 1.2 Flow Cytometry Analysis Software (Beckman Coulter).
8
Immunophenotyping of Regulatory T Cells
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Whole blood, PBMC, and nTreg staining was performed using the FoxP3 Staining Buffer Set purchased from eBioscience (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's recommendations. The following antibodies were used for wholeblood staining: CD14-PacificOrange (Invitrogen, Darmstadt, Germany); CD3-PacificBlue, CD4-AlexaFluor700, CD25-PE (BD Biosciences, Heidelberg, Germany); CD127-APC-AlexaFluor780, CD62L-PerCP-Cy5.5 (BioLegend, San Diego, CA); CD45RA-ECD (Beckman Coulter, Krefeld, Germany); and FoxP3-AlexaFluor488 (BD Pharmingen, Heidelberg, Germany). The following antibodies were used for PBMC stainings: CD3-PE-Cy7, CD4-AlexaFluor700, CD25-PE (BD Biosciences), CD14-PacificOrange (Invitrogen), CD127-APC-AlexaFluor780, CD45RA-ECD (Beckman Coulter); FoxP3-AlexaFluor488, CD137-PE-Cy5 (BD Biosciences), and CD154-PacificBlue (BioLegend). Dead cells were routinely detected by staining with aqua fluorescent reactive dye (Invitrogen). Data acquisition was performed on a FACS LSRII (BD Biosciences) or Navios flow cytometer (Beckman Coulter). FlowJo software V8 (Treestar) was used for further analysis. c l i n i c a l i n v e s t i g a t i o n S Landwehr-Kenzel et al.: nTregs in kidney transplantation nTregs were defined as CD4 þ CD25 high FoxP3 þ cells. By Boolean gating, non-Treg cells were identified and considered Tconv.
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