Total spleen cells from OT-II mice were filtered through a 40μm cell strainer. After red blood cell lysis with ACK buffer, the remaining cells were stained with antibodies targeting CD4 BV711 (Biolegend 100549), CD25 FITC (Biolegend 102005), CD44 AF647 (Biolegend 103017), CD62L PE (Biolegend 104407), TCRb PE-Cy7 (Biolegend 109222), CD11c V450 (BD 560369), MHCII AF700 (Biolegend 107622) and the viability dye eFluor780 (eBioscience 65-0865-14). Naïve T-cells and CD11c+ MHCII+ APCs were FACS-sorted on a FACS aria and subsequently co-cultured at a ratio of 5:1 in the presence of 1 μg/ml OVA peptide 323-339 (EMC BAP-250) and 5ng/ml rmTGF-β1 (R&D Systems, 7666-MB) for 72 hours. Staining of regulatory T-cells and FACS analysis was conducted as described in “Flow cytometric analysis”.
Cd62l pe
Manufactured by BioLegend
Sourced in United States
CD62L-PE is a fluorescent-conjugated antibody that binds to the CD62L (L-selectin) antigen on the surface of cells. CD62L is a cell adhesion molecule involved in the homing and migration of leukocytes. This antibody can be used to detect and quantify CD62L expression on various cell types using flow cytometry or other immunoassay techniques.
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Lab products found in correlation
Cd44 fitc, by BioLegend (3 mentions)
Cd25 fitc, by BioLegend (2 mentions)
Cd4 pe, by BioLegend (2 mentions)
Canto 2, by BD (2 mentions)
Cd4 percp cy5, by BioLegend (2 mentions)
Cd8 brilliant violet 510, by BioLegend (2 mentions)
Cd4 bv711, by BioLegend (1 mentions)
Cd44 af647, by BioLegend (1 mentions)
Tcrb pe cy7, by BioLegend (1 mentions)
Cd11c v450, by BD (1 mentions)
Mhcii af700, by BioLegend (1 mentions)
Efluor 780, by Thermo Fisher Scientific (1 mentions)
Rmtgf β1, by R&D Systems (1 mentions)
Cd3 apc cyanine7, by BioLegend (1 mentions)
Live dead fixable yellow dead cell stain, by Thermo Fisher Scientific (1 mentions)
Cd107b alexa fluor 647, by BioLegend (1 mentions)
Cd8a fitc, by BioLegend (1 mentions)
Ikaros, by Cell Signaling Technology (1 mentions)
Cd3 af488, by BioLegend (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
16 protocols using cd62l pe
1
Regulatory T-cell Induction from Naive T-cells
2
Multi-parameter Immune Cell Profiling
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CD44‐Brilliant Violet 711 (BioLegend, Cat.# 103057, clone: IM7, 1:80), CD8a‐FITC (BioLegend, Cat.# 100706, clone: 53‐6.7, 1:50), CD62L‐PE (BioLegend, Cat.# 104407, clone: MEL‐14, 1:80), CX3CR1‐PE/Cyanine 7 (BioLegend, Cat.# 149015, clone: SA011F11, 1:1333), CD107b‐Alexa Fluor 647 (BioLegend, Cat.# 108512, clone: M3/84, 1:200), CD3‐APC/Cyanine7 (BioLegend, Cat.# 100222, clone: 17A2, 1:80), and LIVE/DEAD Fixable Yellow Dead Cell Stain (Thermo Fisher Scientific, Cat.# L34967, 1:7500).
3
Signaling Pathway Protein Analysis
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AA (ab120916), thapsigargin (ab120286), ORAI3 Ab (ab115558), and NUR77 Ab (ab109180) were purchased from Abcam. Abs recognizing p-CAMKII (Thr286, #12716), p-ERK (Thr202/Tyr204, #4377s), CAMKII (#4436), ERK (#4695), Myb (#12319), AP-2a (#3208), and IKAROS (#9034s) were from Cell Signaling. Abs to β-actin (#A5441) were obtained from Sigma, to p-CD3ζ (Y142) from BD Biosciences (558402) and to CD3ζ from BioLegend (644102). Abs for flow cytometry were purchased from BD Biosciences [CD4-V450 (560345), CD8-PE-Cy7 (557746), CD45RA-AF700 (560673), CD69-Percp-Cy5.5 (560738), p-ERK (Thr202/Try204)-AF670 (561992), and p-SLP76 (Y128)-AF647 (558438)] or BioLegend [CD62L-PE (304806), IKAROS-AF647 (368404), and CD3-AF488 (30045)]. The LIVE/DEADTM Fixable Aqua Dead Cell Stain Kit from Molecular Probes, Thermo Fisher Scientific was used to gate out dead cells; Fluo-8 (21080) and Fura RedTM (F3021) for staining cytoplasmic Ca2+ was from AAT Bioquest, Inc., and Thermo Fisher Scientific, respectively.
4
Flow Cytometry Analysis of CAR T Cell Phenotype
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Expression of EGFR and other proteins on the tumor cell surface was detected by flow cytometry using the anti-EGFR antibody (BD, San Jose, CA, USA). In brief, the CAR T cells were collected from culture and assayed with monoclonal antibodies against human CD25-PC5 and CD69-PC5 (Beckman Coulter Inc., Indianapolis, IN, USA), CD3-PE-CY5, CD4-PE, CD8-FITC, CD45RO-PE-CY5, CD62L-PE, TIM3-PE, and LAG3-Alexa Fluor 647 (Biolegend, San Diego, CA, USA) and CCR7-FITC, CD107α-PE-CY5, and PD-1-PE (BD, San Jose, CA, USA) according to a previous study40 (link) or the manufacturers’ instructions.
EGFR-modified CAR expression was detected by using an indirect method with biotinylated EGFR protein and streptavidin-coupled PE antibody (BD). Fluorescence was assessed using a Beckman Coulter Gallios™ flow cytometer, and the data were analyzed with the FlowJo vX.0.7 and Kaluza v1.5 software (Beckman Coulter Inc.).
EGFR-modified CAR expression was detected by using an indirect method with biotinylated EGFR protein and streptavidin-coupled PE antibody (BD). Fluorescence was assessed using a Beckman Coulter Gallios™ flow cytometer, and the data were analyzed with the FlowJo vX.0.7 and Kaluza v1.5 software (Beckman Coulter Inc.).
5
Splenic T Cell Subsets by FACS
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For splenic T cell sorting, magnetic bead-mediated positively selected CD4+ cells were stained with combinations of antibodies (0.5μg/mL per) to CD62L-PE (BioLegend, San Diego, CA) or CD62L-BB515 (BD Bioscience), CD44-APC (BioLegend, San Diego, CA), CD45Rb-APC/Cy7 (BioLegend, San Diego, CA), CD124 (1μg/mL, BD Biosciences, San Jose, CA), and CD25-BV421 (BD Biosciences, San Jose, CA). For Tregs, FoxP3-RFP reporter signal was used for FACS. Cells were washed twice in MACS buffer (Miltenyi Biotec, San Diego, CA) before sterile cell sorting using a FACSAria (BD Biosciences, San Jose, CA) with the support of the Cytometry & Imaging Microscopy Core Facility of the Case Comprehensive Cancer Center. Analysis of all FACS data was performed using FlowJo v10 (Tree Star, Inc., Ashland, OR).
6
Multiparametric Flow Cytometry of Tumor Immune Cells
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Dissociation of fresh tumor samples and antibody staining was performed as described previously40 (link). Cells were blocked with anti-mouse CD16/CD32 FC block (Biolegend, 1:100) for 10 min on ice and stained with Zombie Aqua Fixable Viability Kit (Biolegend, 1:500) to discriminate live and dead cells. The following antibody cocktails were used: CD4 BUV805 (BD, 1:100), CD3εBUV395 (BD, 1:20), CD8a BV785 (Biolegend, 1:100), CD25 BV650 (Biolegend, 1:50), TCRγ/δ BV421 (Biolegend, 1:100), CD62L PE (Biolegend, 1:500), CD44 APC-Fire (Biolegend, 1:30), CD45 PerCP Cy5.5 (Biolegend, 1:100), CD19 FITC (Biolegend, 1:100), EpCAM APC/AF647 (Biolegend, 1:200) for acquisition of adaptive immune cells; CD11c BUV737 (BD, 1:30), NK1.1 BUV395 (BD, 1:25), Ly6C BV785 (Biolegend, 1:200), CD11b BV650 (Biolegend, 1:100), F4/80 BV421/PB (Biolegend, 1:30), CD45 PerCP Cy5.5 (Biolegend, 1:100), Ly6G PE (Biolegend, 1:200), CD68 APC-CY7 (Biolegend, 1:20), EpCAM APC/AF647 (Biolegend, 1:200) for acquisition of innate immune cells. 1 × 106 events were acquired per antibody panel on the BD LSRFortessa. Flow cytometry data were analyzed using FlowJo software (v10.6.2).
7
Isolation and Characterization of Naive CD4+ T Cells
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For naive CD4 T cell selection, magnetically selected CD4-enriched splenocytes were labeled with CD25-FITC, CD62L-PE, and CD4-PerCP/Cy5.5 antibodies (BioLegend). The cells were then subjected to cell sorting (FACSAria) to obtain CD4+CD62L+CD25− naive T cells. A cell purity of more than 95% was routinely achieved. For cytokine staining, activated T cells were re-stimulated with PMA (50 ng ml−1) and ionomycin (1 μM ) for 4 h and then stained using the Fixation and Permeabilization Solution Kit with BD GolgiStop (BD Biosciences, Franklin Lakes, NJ, USA). For transcription factor staining, the cells were stained with an anti-Foxp3 antibody (eBioscience, Santa Clara, CA, USA) using the Transcription Factor Staining Buffer Set (eBioscience). Occasionally, the FOXP3 Fix/Perm Buffer Set (BioLegend) was used to stain GFP-expressing cells. The cells were then analyzed by flow cytometry (FACSCalibur).
8
Phenotyping and Transgene Detection of CAR-T Cells
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To detect phenotype and subset, CAR-T cells were stained with mouse anti-human CD4 Percp, CD8 PE, CD62L PE, CCR7 Percp and CD45RO PE/Cy7 on day 7 (Biolegend). To determine CAR transgene, Alexa 647-labeled F(ab)2 fragment of Goat anti-Mouse IgG (Jackson ImmunoResearch) was utilized. All samples were analyzed with canto II (BD Biosciences), and data were processed by FlowJo software.
9
Multiparametric Flow Cytometry of Immune Cells
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All blood and processed brain hemi samples were stained using anti-mouse recombinant antibodies: Ly6G BV421 (Biolegend, Clone 1A8), CD45 APC (Biolegend, Clone 30-F11), CD62L PE (Biolegend, Clone MEL-14), and CD11b (Biolegend, Clone M1/70) along with a fixable live/dead stain (Invitrogen L34988). Stained samples were fixed with a 1x fixation buffer (Biolegend 420801) and CountBright™ beads (Invitrogen C36950) were added prior to data acquisition. Fluorescence data was collected by flow cytometry on a Sony SH800S cell sorter. FlowJo (version 10.9.0, BD Biosciences) was used to analyze the data. Cells were gated based on antibody markers and/or scatter. Absolute neutrophil frequencies were calculated for peripheral blood based on the percentage of CountBright™ beads collected.
10
T Cell Phenotyping After EF Stimulation
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For T cell differentiation experiments, T cells were stained 3 days after EF stimulation with anti-human CD45RO-FITC (Immunotools) and CD62L-PE (BioLegend), and the corresponding controls. T cells were washed out of the channel slides, cells were resuspended in 50 μL FACS buffer and incubated with antibodies for 30 min on ice, in the dark. After washing cells with FACS buffer, cells were gated with SSC-A vs. FSC-A for living cells and analyzed for FITC and PE fluorescence by flow cytometry. Quadrants were defined by positive and negative/control PE and FITC signals.
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