For flow cytometric analysis, splenocytes (2 × 105) were labelled with mouse anti-rat monoclonal antibodies (mAb) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridininchlorophylla protein (PercP), allophycocyanin (APC) or APC-Cyanine (Cy)7, as in previous studies60 (link). In this case, the antibodies used were anti-CD4, anti-CD8α, anti-CD8β, anti-TCRαβ, anti-TCRγδ, anti-NKR-P1A, anti-CD25, anti-CD45RA (BD Biosciences, San Diego, USA), anti-CD62L, anti-CD103 (Biolegend, San Diego, CA, USA), anti-TLR-4 (Novus Biologicals, Littlon, CO, USA) and anti-Foxp3 (eBioscience). After staining with standard procedures20 (link), analyses were performed using a GalliosTM Cytometer (Beckman Coulter Inc., Miami, FL, USA) at the CCiT-UB. All results were assessed by the Flowjo v.10 software (TreeStar, Inc., Ashland, OR, USA).
Anti cd62l
Manufactured by BioLegend
Sourced in United States
Anti-CD62L is a lab equipment product that detects the expression of CD62L (L-selectin) on the surface of cells. CD62L is a cell adhesion molecule involved in the homing of lymphocytes to peripheral lymph nodes. This product can be used in flow cytometry analysis to identify and characterize cell populations expressing CD62L.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd44, by BioLegend (17 mentions)
Anti cd4, by BioLegend (16 mentions)
Anti cd3, by BioLegend (14 mentions)
Anti cd45, by BioLegend (12 mentions)
Anti cd11b, by BioLegend (11 mentions)
Anti cd8, by BioLegend (11 mentions)
Anti ifn γ, by BioLegend (6 mentions)
Anti cd11c, by BioLegend (6 mentions)
Anti cd103, by BioLegend (5 mentions)
Anti cd4, by Thermo Fisher Scientific (5 mentions)
Anti ly6g, by BioLegend (5 mentions)
Anti nk1, by BioLegend (5 mentions)
Anti il 10, by BioLegend (4 mentions)
Anti foxp3, by BioLegend (4 mentions)
Anti cd69, by BioLegend (4 mentions)
Anti cd40, by BioLegend (4 mentions)
Anti cd3, by Thermo Fisher Scientific (4 mentions)
Anti cd8α, by BioLegend (4 mentions)
Anti cd19, by BioLegend (4 mentions)
Anti cd80, by BioLegend (4 mentions)
35 protocols using anti cd62l
1
Comprehensive Flow Cytometric Analysis
2
Immunophenotyping Murine Splenocytes
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Spleens were sieved (70 µm), before washing [phosphate-buffered saline (PBS); 350 g, 5 min], re-sieving (40 µm), RBC lysis (eBioscience), washing, and resuspension in FACS buffer (1% BSA, 0.05% NaN3, in PBS) or full RPMI 1640. Cells in FACS buffer were Fc blocked (1:100; BioLegend; 10 min, RT) before staining for T-cell activation with: anti-CD4 (1:800; eBioscience), anti-CD8 (1:100), anti-CD62L (1:80), anti-CD44 (1:400; all BioLegend; 20 min, RT); or for Tregs with: anti-CD4 (1:800), anti-CD25 (1:80; BioLegend; 20 min, RT), before washing, fixation, permeabilization (FOXP3 Fix/Perm; BioLegend), then anti-FOXP3 (1:20; BioLegend; 30 min, RT). Splenocytes in RPMI were treated ±ionomycin/phorbol 12-myristate 13-acetate/brefeldin A (1:500; BioLegend), incubated (5 h, 37°C), washed, resuspended in FACS buffer, Fc blocked, stained with anti-CD4 or anti-CD8 (20 min, RT), washed, fixed, permeabilized (BioLegend), then anti-IL-10, anti-IL-17, and anti-interferon γ (IFNγ; all 1:100; BioLegend; 30 min, RT). For neutrophil, monocyte or Ly6C+ cells, whole blood (ethylenediaminetetraacetic acid) was stained with anti-CD115 (1:100; eBioscience), anti-CD11b (1:800), anti-Ly6G (1:80; both BioLegend), anti-Ly6C (1:400; AbD Serotec, Hercules, CA), at RT for 30 min, before RBC lysis, washing, and analysis by flow cytometry (Accuri C6).
3
Multiparametric Flow Cytometry of Immune Cells
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Spleens were sieved (70 µm), before washing [phosphate-buffered saline (PBS); 350 g, 5
min], re-sieving (40 µm), RBC lysis (eBioscience), washing, and resuspension in FACS
buffer (1% BSA, 0.05% NaN3, in PBS) or full RPMI 1640. Cells in FACS buffer
were Fc blocked (1:100; BioLegend; 10 min, RT) before staining for T-cell activation with:
anti-CD4 (1:800; eBioscience), anti-CD8 (1:100), anti-CD62L (1:80), anti-CD44 (1:400; all
BioLegend; 20 min, RT); or for Tregs with: anti-CD4 (1:800), anti-CD25 (1:80; BioLegend;
20 min, RT), before washing, fixation, permeabilization (FOXP3 Fix/Perm; BioLegend), then
anti-FOXP3 (1:20; BioLegend; 30 min, RT). Splenocytes in RPMI were treated
±ionomycin/phorbol 12-myristate 13-acetate/brefeldin A (1:500; BioLegend), incubated (5 h,
37°C), washed, resuspended in FACS buffer, Fc blocked, stained with anti-CD4 or anti-CD8
(20 min, RT), washed, fixed, permeabilized (BioLegend), then anti-IL-10, anti-IL-17, and
anti-interferon γ (IFNγ; all 1:100; BioLegend; 30 min, RT). For neutrophil, monocyte or
Ly6C+ cells, whole blood (ethylenediaminetetraacetic acid) was stained with
anti-CD115 (1:100; eBioscience), anti-CD11b (1:800), anti-Ly6G (1:80; both BioLegend),
anti-Ly6C (1:400; AbD Serotec, Hercules, CA), at RT for 30 min, before RBC lysis, washing,
and analysis by flow cytometry (Accuri C6).
min], re-sieving (40 µm), RBC lysis (eBioscience), washing, and resuspension in FACS
buffer (1% BSA, 0.05% NaN3, in PBS) or full RPMI 1640. Cells in FACS buffer
were Fc blocked (1:100; BioLegend; 10 min, RT) before staining for T-cell activation with:
anti-CD4 (1:800; eBioscience), anti-CD8 (1:100), anti-CD62L (1:80), anti-CD44 (1:400; all
BioLegend; 20 min, RT); or for Tregs with: anti-CD4 (1:800), anti-CD25 (1:80; BioLegend;
20 min, RT), before washing, fixation, permeabilization (FOXP3 Fix/Perm; BioLegend), then
anti-FOXP3 (1:20; BioLegend; 30 min, RT). Splenocytes in RPMI were treated
±ionomycin/phorbol 12-myristate 13-acetate/brefeldin A (1:500; BioLegend), incubated (5 h,
37°C), washed, resuspended in FACS buffer, Fc blocked, stained with anti-CD4 or anti-CD8
(20 min, RT), washed, fixed, permeabilized (BioLegend), then anti-IL-10, anti-IL-17, and
anti-interferon γ (IFNγ; all 1:100; BioLegend; 30 min, RT). For neutrophil, monocyte or
Ly6C+ cells, whole blood (ethylenediaminetetraacetic acid) was stained with
anti-CD115 (1:100; eBioscience), anti-CD11b (1:800), anti-Ly6G (1:80; both BioLegend),
anti-Ly6C (1:400; AbD Serotec, Hercules, CA), at RT for 30 min, before RBC lysis, washing,
and analysis by flow cytometry (Accuri C6).
4
Comprehensive NK Cell Phenotyping
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The following fluorescently conjugated antibodies were used for phenotypic analysis of NK cells: anti-LFA-1 (363404); anti-CXCR3 (353720); anti-PD-1 (329906); anti-CD107a (328612); anti-NKP44 (325116); anti-CD158e1 (312706); anti-CD158d (347006); anti-CD27 (356412); anti-CCR4 (359412); anti-DNAM-1 (338316); anti-CD16 (302040); anti-Granzyme B (515406); anti-CD62L (304806); anti-CD69 (310910); anti-NKP80 (346706); anti-NKP30 (325210); anti-CD158f (341304); anti-CD158b (312612); anti-Tim-3 (345012); anti-CD94 (305504); anti-TIGIT (372706); anti-TRAIL (308206); anti-CD57 (322306); anti-CX3CR1 (341610); anti-NKG2D (320808); anti-Perforin (353310); anti-Ki67 (350504); anti-IFN-γ (506518); anti-CD94 (305506); anti-NKp46 (331916); anti-CTLA4 (369614); anti-CD96 (338416); anti-41BB (309818); anti-CD25 (356108) were purchased from Biolegend. anti-CD159a/NKG2A (FAB1059P) and anti-NKG2C (FAB138G) were purchased from R&D.
The following fluorescently conjugated antibodies were used to identify immune cell types in eNK or PBMC: anti-human Lineage Cocktail (348803); anti-CD56 (362550); anti-CD3 (300430); anti-CD33 (366620); anti-HLA-DR (307606); anti-CD14 (301836); anti-CD19 (302242); anti-CD11b (301322); anti-CD25 (356108); and anti-FOXP3 (320108) were purchased from Biolegend, San Diego, CA, USA.
The following fluorescently conjugated antibodies were used to identify immune cell types in eNK or PBMC: anti-human Lineage Cocktail (348803); anti-CD56 (362550); anti-CD3 (300430); anti-CD33 (366620); anti-HLA-DR (307606); anti-CD14 (301836); anti-CD19 (302242); anti-CD11b (301322); anti-CD25 (356108); and anti-FOXP3 (320108) were purchased from Biolegend, San Diego, CA, USA.
5
Multicolor Flow Cytometry Panel for Immune Profiling
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Cells were stained with the following antibodies obtained from BD Biosciences (Franklin Lakes, NJ, USA), eBioscience, or BioLegend (San Diego, CA, USA): anti-CD4 (BioLegend, 100451), anti-CXCR3 (eBioscience, 12-1831-82), anti-CCR7 (eBioscience, 12-1971-82), anti-CD44 (eBioscience, 12-0441-83), anti-CD62L (BioLegend, 104406), anti-CD11c (BD Bioscience, 553801), anti-MHCII (BioLegend, 107631), anti-CD80 (BD Biosciences, 553769), anti-CD86 (BD Biosciences, 553692), anti-B220 (eBioscience, 12-0452-83), anti-CD8 (BD Bioscience, 553032), anti-CD103 (BD Bioscience, 557495), anti-CD45 (BioLegend, 103132), and anti-CD11b (BioLegend, 101263). For Th1 and Treg analyses, cells were stained for surface markers, permeabilized with the Intracellular Fixation and Permeabilization Buffer Set (eBioscience, 88-8824-00), and then stained with anti-IFN-γ (BioLegend, 505825) and anti-FOXP3 (eBioscience, 17-5773-82) antibodies. The following antibodies were obtained from Cell Signaling Technology (Danvers, Massachusetts, USA): anti-Ezh2 (#5246), anti-Runx1 (#4336), and anti-H3K27me3 (#9733). The Alexa Fluor™ 488 Goat Anti-Rabbit SFX Kit from Invitrogen (Carlsbad, CA, USA) was used as a secondary antibody. Multicolor flow cytometric analysis was performed using a CytoFLEX LX (Beckman Coulter, Indianapolis, IN, USA).
6
Tumor-infiltrating Lymphocyte Characterization and Profiling
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Tumor-infiltrating lymphocyte isolation and other procedures, including stimulation, fixation, permeabilization, and intracellular staining, were described previously [39 (link)]. Antibodies used for surface marker staining included anti-CD45 (BioLegend, clone: 30-F11), anti-CD3 (BioLegend, clone: 17A2), anti-CD11c (BioLegend, clone: N418), anti-MHC-II (Invitrogen, clone: M5/114.15.2), anti-CD11b (BioLegend, clone: M1/70), anti-F4/80 (BioLegend, clone: BM8), anti-B220 (BioLegend, clone: RA3-6B2), anti-CD4 (BD Horizon, clone: RM4-5), anti-CD8 (BD Pharmingen, clone: 53-6.7), anti-CD40 (BioLegend, clone: 3/23), anti-CD40L (BioLegend, clone: SA047C3), anti-IL2Rα (BioLegend, clone: 3C7), and anti-CD44 (BioLegend, clone: IM7), and anti-CD62L (BioLegend, clone: MEL-14) antibodies. For cytokine staining, an anti-IFN-γ antibody (BD Pharmingen, clone: XMG1.2) was used after TIL fixation and permeabilization. Data were acquired on a BD FACSCanto II flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).
7
Immune Cell Profiling of Pancreatic Islets
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Mouse islet cells were stained with anti-CD45 and biotinylated anti-H2Kd (BD Pharmingen) followed by streptavidin-allophycocyanin (BioLegend). β‐cells were identified by autofluorescence (23 (link)). Dead cells were excluded using propidium iodide. The immune cells infiltrating islets and spleens were stained with anti-CD45, anti-CD3, anti-CD4, anti-CD8a, anti-CD44, anti-CD62L, anti-KLRG-1, anti-CD11b, anti-Ly6G (all BioLegend), anti-BrdU, anti-pSTAT3, and anti-pSTAT5 (all BD Bioscience). Data were collected on the FACS Fortessa cell analyzer (BD Bioscience, San Jose, CA, USA) and were analyzed using FlowJo Software version 10 (TreeStar, Inc, Ashland, OR, USA).
8
Multiparametric Flow Cytometry of Immune Cells
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Different panels of antibody cocktails were generated from anti‐CD45 (BioLegend, 103116, 30‐F11), anti‐CD3 (BioLegend, 100218, 17A2), anti‐CD4 (BioLegend, 100421, GK1x.5), anti‐CD8a (BioLegend, 100711, 53‐6.7), anti‐NKp46 (BioLegend, 137606, 29A1.4), anti‐CD11c (BioLegend, 117307, N418), anti‐CD25 (Biolegend, 101908, 3C7), anti‐FOXP3 (Biolegend, 126404, MF‐14), anti‐granzyme B (BioLegend, 372208, QA16A02), anti‐IFNγ (BioLegend, 505849, XMG1.2), anti‐IFNγ (BioLegend, 505806, XMG1.2, anti‐CD62L (BioLegend, 104432, MEL‐14) and anti‐CD44 (BD Biosciences, 560568, IM7) antibodies, and the AmCyan Live/Dead Cell Staining Kit (Thermo Fisher Scientific). All antibodies were diluted at optimized dilutions before being used.
Female 7–8 weeks old C57BL/6 mice (n = 4–5 per group) were intravenously administered with the equivalent dose (3.8 × 1010 vg/mouse) of either free AAV‐fLuc or RBC‐AAV‐fLuc. Mice were then euthanized on day 31, and spleen and lungs were collected. Organ dissociation kits (Miltenyi Biotec) were used per manufacturer's instruction to generate a single‐cell suspension from excised organs, and cells were stained with the antibodies listed above and analyzed using flow cytometry (BD LSR II Analyser). Flow cytometry data analyses were performed using FlowJo 10 software.
Female 7–8 weeks old C57BL/6 mice (n = 4–5 per group) were intravenously administered with the equivalent dose (3.8 × 1010 vg/mouse) of either free AAV‐fLuc or RBC‐AAV‐fLuc. Mice were then euthanized on day 31, and spleen and lungs were collected. Organ dissociation kits (Miltenyi Biotec) were used per manufacturer's instruction to generate a single‐cell suspension from excised organs, and cells were stained with the antibodies listed above and analyzed using flow cytometry (BD LSR II Analyser). Flow cytometry data analyses were performed using FlowJo 10 software.
9
Liver Leukocyte Isolation and Flow Cytometry
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Leukocytes were isolated from the liver in the way as described previously40 (link). For flow cytometry, cells were stained for surface antigens with anti-NK1.1, anti-CD3, anti-CD4, anti-CD8α, anti-CD19, anti-CD44, anti-CD62L (BioLegend, San Diego, CA), or/and for intracellular antigens with anti-IFN-γ, anti-IL-4, anti-IL-17A, anti-TNF-α and anti-IL-10 (BioLegend). IgG isotype controls (Biolegend) were used in parallel. To detect Treg cells, cells were stained with anti-NK1.1, anti-CD3, anti-CD4, anti-CD25 at 4 °C for 30 minutes; and incubated with anti-mouse Foxp3 antibodies according to the manufacturer’s instructions (eBioscience). Methods were the sameas those described previously9 (link).
10
Establishment of Murine Tumor Models
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