Mouse spleens were homogenized, and single-cell suspensions were prepared and counted after lysis with RBC lysis buffer (BioLegend). For macrophage staining, 1 × 106 cells were incubated with FITC-F4/80 (Biolegend) and APC-Ly6c (Biolegend) antibodies at room temperature for 30 min. The cells were rinsed with 1 ml PBS and resuspended with 200 μl PBS for detection. For the detection of Tcm (central memory T cell) and Tem (effector memory T cell), 1 × 106 cells were incubated with FITC-CD62L (Biolegend), PE-CD44 (Biolegend), Percp-cy5.5-CD4 (Biolegend), and APC-CD8 (Biolegend) antibodies for 30 min. The cells were then subjected to 1 ml PBS washing, followed by suspending with 200 μl PBS for cytometry detection. For intracellular staining, 2 × 106 cells were stimulated with PMA plus ionomycin (Invitrogen) for 5 hr. After incubation with FITC-CD4 (Biolegend) and PE-CD8 (Biolegend) antibodies at room temperature for 30 min, the cells were then incubated with Fix/Perm according to kit instructions and APC-IFN-γ (Biolegend) antibodies for 30 min. Then the cells were rinsed with 1 ml PBS and resuspended with 200 μl PBS for detection.
Cd62l fitc
Manufactured by BioLegend
Sourced in United States
CD62L-FITC is a fluorochrome-conjugated antibody that specifically binds to the CD62L (L-selectin) cell surface antigen. CD62L is a cell adhesion molecule involved in the homing of lymphocytes to peripheral lymph nodes. The FITC fluorescent label allows for the detection and analysis of CD62L-expressing cells using flow cytometry.
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Lab products found in correlation
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Ifn γ pe, by BioLegend (2 mentions)
Ifn γ fitc, by BioLegend (2 mentions)
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Cytofix cytoperm kit, by BD (2 mentions)
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F4 80 fitc, by BioLegend (1 mentions)
Rbc lysis buffer, by BioLegend (1 mentions)
Cd4 apc, by BioLegend (1 mentions)
Cd45 pe cy7, by Thermo Fisher Scientific (1 mentions)
14 protocols using cd62l fitc
1
Multiparameter Immune Cell Analysis
2
Multiparameter Flow Cytometry Immunophenotyping
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The surface markers included FITC‐CD3 (MA1‐7640, eBioscience, San Diego, CA, USA), APC‐Cy7‐CD4 (100413, Biolegend, San Diego, CA, USA), APC‐CD4 (100411, Biolegend), PerCP‐CD8 (100,731, Biolegend), PE‐Cy7‐CD8 (100721, Biolegend), PE‐Foxp3 (12‐5773‐82, eBioscience), APC‐Helios (17‐9883‐42, eBioscience), PerCP‐CD44 (103035, Biolegend), FITC‐CD62L (104405, Biolegend), FITC‐lin (22‐7770‐72, eBioscience), PerCP‐CD34 (50‐0341‐82, eBioscience), APC‐Sca‐1 (17‐5981‐82, eBioscience), PE‐Sca‐1 (12‐5981‐82, eBioscience), APC‐c‐kit (17‐1171‐82, eBioscience), PE‐c‐kit (12‐1171‐82, eBioscience), PE‐B220 (12‐0452‐82, eBioscience), FITC‐IgM (11‐5790‐81, eBioscience), PE‐Cy7‐CD19 (12‐0193‐82, eBioscience), APC‐CD19 (17‐0193‐82, eBioscience), PE‐Cy7‐CD16/32 (25‐0161‐82, eBioscience), PE‐Cy7‐CD45 (25‐0451‐82, eBioscience), PerCP‐5.5‐CD45.2 (45‐0454‐82, eBioscience), and APC‐Cy7‐CD45.1 (17‐0453‐82, eBioscience) obtained from Invitrogen. Intracellular staining with PE‐Foxp3 (72‐5775‐40, eBioscience) was performed with Foxp3 staining kits (Invitrogen, Carlsbad, CA, USA).
3
Tumor Immune Cell Profiling by Flow Cytometry
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Tumors were processed for flow cytometry into single cells using mechanical and enzymatic digestion (enzyme mixture—collagenase, DNAse, and hyaluronidase). Red blood cells were lysed as described above and viable tumor cells counted. The following antibodies were used to stain immune cell populations: Ghost violet 510 Live/Dead, Pacific Blue CD45, PE-594 CD3, PE/Cy7 CD8, APC/Cy7 CD4, BV605 PD-1, APC Tim3, FITC CD62L, BV711 CD44 (BioLegend).
Samples were run on an LSR Fortessa machine. Single color compensation controls were performed using compensation beads (Thermo Fisher Scientific, Waltham, MA, USA) to correct for overlap in signal among antibodies. Spleen samples were used to set a gating strategy for CD3+/CD4+ and CD3+/CD8+ T cells. FlowJo software (version 10) was used to perform all downstream analyses on subpopulations.
Samples were run on an LSR Fortessa machine. Single color compensation controls were performed using compensation beads (Thermo Fisher Scientific, Waltham, MA, USA) to correct for overlap in signal among antibodies. Spleen samples were used to set a gating strategy for CD3+/CD4+ and CD3+/CD8+ T cells. FlowJo software (version 10) was used to perform all downstream analyses on subpopulations.
4
Generating and Staining pMHC Multimers
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pMHC monomers were generated as previously described66 (link). All pMHC-multimer reagents were generated by 45 min incubation of 4 μg biotinylated pMHC monomer with 1 μg streptavidin-BV421 (Biolegend, reference #405225) or Streptavidin-APC (eBioscience, reference #17-4317-82) in a total volume of 100 μl FACS buffer for staining of up to 1x107 cells. pMHC-multimer staining was performed separately from surface antibody staining for 45 min on ice. Surface antibody staining was performed for 25 min on ice subsequent to pMHC-multimer staining. Depending on the respective experiments, a subset of the following antibodies were used: anti-human TCR α/β FITC (BioLegend, reference #306706), CD3 PC7 (Beckman Coulter, reference #737657), CD8 PE (Invitrogen, reference #MHCD0804), anti-murine TCR β-chain APC (BioLegend, reference #109212), anti-murine TCR β-chain APC/Fire750 (BioLegend, reference #109246), CD62L FITC (Biolegend, reference #304804), CD45-RO PC7 (BioLegend, reference #304230), Lag3 FITC (Biolegend, reference #369308) and PD-1 APC (Invitrogen, reference #17-2799-42). Live/dead discrimination was performed with propidium iodide (LifeTechnologies, reference #P1304MP).
5
Polychromatic Flow Cytometry for Treg and Th Subsets
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Regulatory T cells (Treg) were defined as CD4+CD25+CD127low/- cells. Other CD4+T subsets were identified by differential expression of CCR4, CXCR3, and CCR6 as previously reported (7 (link), 9 (link)): CXCR3+CCR4−CCR6− (Th1), CXCR3−CCR4+CCR6− (Th2), CXCR3−CCR4+CCR6+(Th17), and CXCR3+CCR4−CCR6+ (Th1Th17) (Figure 1
The fluorochrome-conjugated antibodies used for polychromatic flow cytometry analysis were CD3-BV510 (Biolegend, USA, catalog no. 317332), CD4-APC-Cy7 (Biolegend, USA, catalog no. 317418), CCR4-APC (Biolegend, USA, catalog no. 359404), CXCR3-PE (Biolegend, USA, catalog no. 353706), CCR6-PE-Cy7 (Biolegend, USA, catalog no. 353418), CD25-BV421 (Biolegend, USA, catalog no. 302630), CD127-FITC (Biolegend, USA, catalog no. 351312), PE-Cy7-CCR7 (Biolegend, USA, catalog no. 353226) and CD62L-FITC (Biolegend, USA, catalog no. 304804). A viability dye, 7-AAD (Becton Dickinson, USA, catalog no. 559925), was used to exclude dead cells. Isotype antibodies were also used as negative controls for every detection to set proper gating for the receptor expression. Cells were analyzed by fluorescence-activated cell sorting (FACS), using the BD LSRII cytometer and FlowJo software.
The fluorochrome-conjugated antibodies used for polychromatic flow cytometry analysis were CD3-BV510 (Biolegend, USA, catalog no. 317332), CD4-APC-Cy7 (Biolegend, USA, catalog no. 317418), CCR4-APC (Biolegend, USA, catalog no. 359404), CXCR3-PE (Biolegend, USA, catalog no. 353706), CCR6-PE-Cy7 (Biolegend, USA, catalog no. 353418), CD25-BV421 (Biolegend, USA, catalog no. 302630), CD127-FITC (Biolegend, USA, catalog no. 351312), PE-Cy7-CCR7 (Biolegend, USA, catalog no. 353226) and CD62L-FITC (Biolegend, USA, catalog no. 304804). A viability dye, 7-AAD (Becton Dickinson, USA, catalog no. 559925), was used to exclude dead cells. Isotype antibodies were also used as negative controls for every detection to set proper gating for the receptor expression. Cells were analyzed by fluorescence-activated cell sorting (FACS), using the BD LSRII cytometer and FlowJo software.
6
Flow Cytometry Analysis of Lymphocytes
7
Comprehensive Treg Phenotyping by Flow Cytometry
8
Comprehensive Immune Cell Profiling
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The cells were extracted from lymph nodes, spleen, or colon tissue digested by collagenase. Isolated cells were stained with following antibodies: CD8-PE-CY7 (Thermo Scientific), CD4-APC-Fire™ 750 (BioLegend), IFN-γ-FITC (BioLegend), IL-17-APC (BioLegend), FOXP3-PE (BioLegend), CD19-PE (BioLegend), NK1.1-FITC (BioLegend), CD11B-APC (BioLegend), Gr-1-FITC (BioLegend), F4/80-PE (BioLegend), CD11C-APC-CY7 (BioLegend), MHC-II-PE/Dazzle™ 594 (BioLegend), CD8-APC (BioLegend), Ki67-PerCP-CY5.5 (BioLegend), Annexin V-FITC (BioLegend), CD103-PE (BioLegend), CD69-PE-CY7 (BioLegend), CD44-PE-CY7 (BioLegend), CD62L-FITC (BioLegend), CD107-FITC (BioLegend), IFN-γ-APC (BioLegend), CD107-PE (BioLegend), IFN-γ-PE (BioLegend), CD3-FITC (BioLegend), CD3-APC/Fire™ 750 (BioLegend), CD8-APC (BioLegend), IFN-γ-PE-CY7 (BioLegend), CD69-APC/Fire™ 750 (BioLegend), Tetramer-SIINFEKL-PE (MBL), p-JNK-PE (Cell Signaling Technology), p-IRAK4-Alexa Fluor 488 (Cell Signaling Technology). Intracellular staining was carried out using the Cytofix/Cytoperm kit (BD Pharmingen) following a 4 h restimulation by PMA/ionomycin (Sigma) in the presence of GolgiPlug (BD Pharmingen). Flow cytometric data acquisition was performed on a NovoExpress flow cytometer and analyzed with NovoExpress software (ACEA Biosciences).
9
Profiling CMV- and IAV-specific CD8+ T cells
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The procedure of flow cytometric analysis of CMV- and IAV-specific CD8+ T cells prior to and post in vitro stimulation was described [40 (link)]. Briefly, freshly PMBCs were stained with fluorescently labeled dextramers or tetramers specific to CMV-pp65 (NLVPMVATV) and IAV-M1 (GILGFVFTL) (Immudex, Copenhagen, Denmark and NIAID tetramer core) first at room temperature for 20 min; followed by staining of cell surface markers including antibody against CD3, CD8, CD62L, CD45RA, CD95, CD27, CD28 CD70, CD127, and CD69 at 4°C for 30 min. Cells were washed again with FACS buffer (Hanks solution with 0.3% Sodium Azide), and then fixed immediately with 3% formaldehyde and 1% FBS in FACS buffer. Fixed cells were further stained with intracellular markers (perforin and granzyme B) at 4°C for 30 min. Stained cells were collected by BD_Symphony and were analyzed using FlowJo version 7.6.5 software.
Antibodies (CD3-BV570, CD62L-FITC, CD62L-PE-Cy7, CD45RA-PE-Cy7, CD45RA-APC, CD95-PE-Cy5, CD27-PE, CD28-BV785, CD28-PerCP-Cy5.5, CD70-PE, CD127--BV711, CD69-BV650, and granzyme B) were purchased from Biolegend, and antibodies against CD8-BUV496, CD27-BUV395, and perforin-PE-CF594 were purchased from BD.
Antibodies (CD3-BV570, CD62L-FITC, CD62L-PE-Cy7, CD45RA-PE-Cy7, CD45RA-APC, CD95-PE-Cy5, CD27-PE, CD28-BV785, CD28-PerCP-Cy5.5, CD70-PE, CD127--BV711, CD69-BV650, and granzyme B) were purchased from Biolegend, and antibodies against CD8-BUV496, CD27-BUV395, and perforin-PE-CF594 were purchased from BD.
10
Detailed Peptide-Stimulated T Cell Profiling
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Cells isolated from spleen, lungs or peripheral blood were cultured with 2 µg/ml of two immunodominant peptides (Pepscan, Lelystad, The Netherlands), [SSTHEANTMAMMARDT] and [AGYAGTLQSLGAEIAV] of the TB10.4 protein, previously demonstrated to stimulate both CD4+ & CD8+ T cell responses (Kaveh & Hogarth, unpublished); 1 µg/ml anti-CD28 (BD Biosciences) and 10 µg/ml Brefeldin A (Sigma) for 16 h at 37 °C/5% CO2. Cells were washed at 300 g for 5 min and surface stained with combinations of CD62L-FITC, CD27-PerCP-Cy5.5, CD8-AF700, CD44-BV421, CD127-PE-Cy7, CD69-FITC, CCR7-BV421, live/dead-Zombie Aqua (all BioLegend) and CD4-APC-H7 (BD Biosciences). Cells were then washed, treated with BD Biosciences Cytofix/Cytoperm as per manufacturer’s instructions and stained intracellularly with combinations of IFN-γ-PE-Cy7, IL-2-APC (both eBioscience), IFN-γ-BV605 and TNF-α-BV605 (both BioLegend). Cells were washed again and analysed using an LSRFortessa™ analyser utilising a 532 nm laser for PE and PE-conjugate excitation with FACSDiva™ software (BD Biosciences). Final analysis was performed using FlowJo® software (Tree Star Inc.) on a minimum of 100,000 live lymphocytes (50,000 for peripheral blood).
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