Lymphocytes from fresh livers and spleens were prepared essentially as previously reported.26 (link) Briefly, excised liver from recipient mice was passed gently through a 200 μm steel mesh using a sterile syringe plunger. The liver cell suspension was collected, washed in PBS, and centrifuged for 10 min at 500 × g. The pellet was resuspended in 40% Percoll (GE Healthcare). The cell suspension was gently overlaid onto 70% Percoll and centrifuged for 25 min at 750 × g. Mononuclear cells were collected from the interface. Splenic cells were obtained from homogenized splenic tissue and filtered through a sieve. Contaminating red blood cells were lysed using red blood cells lysis buffer (BioLegend) and washed twice in PBS. Single cell suspensions obtained from the spleen and liver were subjected to flow cytometry analysis to detect surface antigens. The antibodies used to identify specific antigens were anti-CD3 (17A2), anti-CD4 (GK1.5), anti-CD8a (53-6.7), anti-CD44 (IM7), and anti-CD62L (MEL-14; all from BioLegend). Stained samples were assessed on a BD Accuri C6 Flow Cytometer (Becton Dickinson) and data analyzed using FlowJo Software (Tree Star).
Anti cd62l mel 14
Manufactured by BioLegend
Sourced in Germany, United States
Anti-CD62L (MEL-14) is a monoclonal antibody that recognizes the mouse L-selectin (CD62L) protein. L-selectin is a cell adhesion molecule expressed on the surface of various leukocytes, including lymphocytes, monocytes, and neutrophils.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd44 im7, by BioLegend (5 mentions)
Anti cd4 rm4 5, by Thermo Fisher Scientific (3 mentions)
Anti cd8a 53 6, by BioLegend (3 mentions)
Anti cd44 im7, by BD (3 mentions)
Anti cd25 pc61, by Thermo Fisher Scientific (2 mentions)
Anti ctla 4 uc10 4b9, by BioLegend (2 mentions)
Anti cd3 17a2, by BioLegend (2 mentions)
Anti cd25 pc61, by BioLegend (2 mentions)
Anti cd4 rm4 5, by BioLegend (2 mentions)
Anti il 4 11b11, by BioLegend (2 mentions)
Anti il 13 ebio13a, by Thermo Fisher Scientific (2 mentions)
Anti cd4 gk1, by BioLegend (2 mentions)
Percoll, by GE Healthcare (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Red blood cells lysis buffer, by BioLegend (1 mentions)
Anti nk1.1 pk136, by BioLegend (1 mentions)
Anti f4 80 bm8, by BioLegend (1 mentions)
Anti cd28 37, by BioLegend (1 mentions)
Ki67 b56, by BD (1 mentions)
Anti mhc class 2 m5 114, by BD (1 mentions)
15 protocols using anti cd62l mel 14
1
Isolation and Analysis of Hepatic and Splenic Lymphocytes
2
Multiparametric Flow Cytometry Analysis
3
GVHD Induction and Treg Modulation
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BALB/c mice were conditioned with total body irradiation (2x400 cGy, 200 kV X-ray source; Kimtron), injected with 5x106 T-cell depleted bone marrow (TCD-BM) cells and 5x105 purified Tregs at day 0 followed by 1x106 CD4+/CD8+ conventional T cells (Tcons) from C57BL/6 or luc+ C57BL/6 mice at day +2. If stated, Tregs were incubated with purified Anti-CD62L (Mel14; BioLegend) for 1 hour at 4°C prior to injection.
4
Analysis of Tumor-Specific T-Cell Responses
5
Comprehensive Immune Cell Profiling
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Samples of tumor, spleen, lymph node, and blood were harvested after killing the mice. Single-cell suspensions of tumors were obtained using a Tumor Dissociation Kit (Miltenyi). The single cells were then incubated in MACS buffer (PBS supplemented with 2% FBS and 1 mM ethylenediaminetetraacetic acid [EDTA]) containing 10 µg/mL CD16/CD32 antibody (2.4G2, BD PharMingen) for 30 min at 4 °C and then stained with the antibodies. Staining reagents included anti-CD45 (30-F11, BioLegend), anti-CD45 (30-F11, BioLegend), anti-CD3 (17A2, BioLegend), anti-CD3 (17A2, BioLegend), anti-CD4 (RM4-5, BioLegend), anti-CD8a (53-6.7, BioLegend), anti-CD25 (PC61, BioLegend), anti-CD44 (IM7, BioLegend), and anti-CD62L (MEL-14, BioLegend). Data were collected on an BD FACSAria Fusion flow cytometer and analyzed with FlowJo software (TreeStar). Dead cells and cell aggregates were excluded from analyses based on a LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (eBioscience), or DAPI, with forward scatter/side scatter characteristics. The immune cell gating strategy was as follows: T cell: CD45+ CD3+, CD4+ T cell: CD45+ CD3+ CD4+, CD8+ T cell: CD45+ CD3+ CD8+, B cell: CD45+ CD3− CD19+.
6
Multiparametric Flow Cytometry Analysis
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Cell staining was performed using the following monoclonal antibodies: anti-CD4 (RM4-5; eBioscience), anti-CD25 (PC61.5; eBioscience), anti-CD44 (IM7; BioLegend), and anti-CD62L (MEL-14; BioLegend). For intracellular staining, cells were first re-stimulated with a cell stimulation cocktail (00-4975-03; eBioscience) for 4 h at 37°C, after which staining of cell surface markers was performed. After staining of the surface markers, the cells were fixed and permeabilized. Intracellular staining was performed using the following monoclonal antibodies: anti-IL-17A (eBio17B7; eBioscience), anti-IFN-γ (XMG1.2; eBioscience), anti-IL-4 (11B11; BioLegend), anti-IL-13 (eBio13A; eBioscience), and anti-Foxp3 (FJK-16s; eBioscience). Stained cells were detected by flow cytometry (FACSCanto II; BD Biosciences), and data were analyzed using FlowJo software version 10.0.7 (Tree Star).
7
Lymphocyte Immunophenotyping by Flow Cytometry
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Peripheral lymph nodes and spleens from indicated mice were grinded into single cells. Cells were washed in phosphate-buffered saline containing 2% (w/v) fetal bovine serum and then stained with indicated antibodies for analysis of surface proteins. For intracellular proteins, cells were fixed and permeabilized with Pharmingen Transcription Factor Buffer Set (BD Pharmingen, 562574). Flow cytometry was performed on CytoFLEX LX (Beckman).
Antibodies used were listed as follows: anti-CD4 (GK1.5, eBioscience), anti-CD8α (53-6.7, BD Pharmingen), anti-CD44 (IM7, BioLegend), anti-CD62L (MEL-14, BioLegend), and anti-Foxp3 (150D, BioLegend).
Antibodies used were listed as follows: anti-CD4 (GK1.5, eBioscience), anti-CD8α (53-6.7, BD Pharmingen), anti-CD44 (IM7, BioLegend), anti-CD62L (MEL-14, BioLegend), and anti-Foxp3 (150D, BioLegend).
8
Flow Cytometric Profiling of T Cell Subsets
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For flow cytometric analysis of surface proteins, 1 × 106 cells were stained with a 1:100 antibody diluted in PBS for 20 min at 4 °C. The following antibodies were used: anti-human IgG-F(ab’)2 (polyclonal, Jackson ImmunoResearch, Ely, UK), anti-CD69 (H1.2F3, Becton Dickinson, Heidelberg, Germany), anti-CD127 (SB/199, BioLegend, Koblenz, Germany), anti-CD62L (MEL-14; BioLegend), anti-CTLA4 (UC10-4B9, BioLegend), anti-GITR (DTA-1, eBioscience, Frankfurt, Germany), anti-CD25 (PC61, Becton Dickinson), anti-CD4 (RM4-5, BioLegend), anti-Thy1.1 (OX-7, BioLegend), and anti-CD8a (53-6.7; BioLegend). Fixable viability dye (eBioscience) was used. For intracellular staining, a fixation/permeabilization kit (eBioscience) and anti-Foxp3 (MF-14, BioLegend) antibodies were used (1:100, 30 min, RT).
For phenotypic analysis, a fluorescence minus one (FMO) control was performed for each marker. After measurement with the flow cytometer, cells were gated either on their Thy1.1 (for cTregs) or on their CD4 expression (nTregs). The histograms were normalized to the mode.
For phenotypic analysis, a fluorescence minus one (FMO) control was performed for each marker. After measurement with the flow cytometer, cells were gated either on their Thy1.1 (for cTregs) or on their CD4 expression (nTregs). The histograms were normalized to the mode.
9
Multiparameter Flow Cytometry Analysis
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Cells were washed with PBS containing 0.5% BSA and incubated for 30 minutes on ice with the following antibodies: anti-CD4 (RM-4.5; eBioscience), anti-CD8 (53.6.7; BD), anti-CD44 (IM.7; BD), anti-CD25 (PC61; BD), anti-CD62L (MEL14; Biolegend), anti-TCRβ (H57-597; eBioscience), anti-TCRγδ (BD), anti-CD69 (H1.2F3; eBioscience), anti-CD11b (M1/70; BD), anti-Gr1 (RB6-8C5; eBioscience) and anti-HSA (M1/69, eBioscience). Cells were then washed in PBS with 0.5% BSA and data was collected using a Fortessa cytometer (BD Bioscience) and analyzed using FlowJo software (Treestar, Ashland, OR).
In the case of intracellular staining, cells were fixed and permeabilized using the Foxp3 buffer staining kit (eBioscience) according to the manufacturer’s instructions prior to staining for intracellular Foxp3 expression using an anti-Foxp3 antibody (FJK-16s, BD), for 30 minutes.
For Annexin V staining, cells were washed with PBS and stained with BD Pharmingen Annexin V Apoptosis Detection Kit I according to the manufacturer’s instructions.
The detection of BrdU was performed using BD Pharmingen BrdU Flow Kit according to the manufacturer’s instructions.
In the case of intracellular staining, cells were fixed and permeabilized using the Foxp3 buffer staining kit (eBioscience) according to the manufacturer’s instructions prior to staining for intracellular Foxp3 expression using an anti-Foxp3 antibody (FJK-16s, BD), for 30 minutes.
For Annexin V staining, cells were washed with PBS and stained with BD Pharmingen Annexin V Apoptosis Detection Kit I according to the manufacturer’s instructions.
The detection of BrdU was performed using BD Pharmingen BrdU Flow Kit according to the manufacturer’s instructions.
10
Immune Cell Profiling of Tumor Samples
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