C57Bl/6 mice (Thy1.2+) were challenged with DENV1, JEV, and YFV (five mice per group) by intraperitoneal injection with 1 × 106 PFU or injected with saline. Five weeks after infection, the spleens were collected and the total T cells were purified by magnetic column using the Pan T Cell Isolation Kit II (mouse; MACS Miltenyi Biotec). C57Bl/6 mice (Thy1.1+) received an adoptive transfer by tail vein injection of 1 × 106 total T cells per mouse, with either T cells purified from mice exposed to DENV1, YFV, JEV, or saline. The mice were challenged with DENV1 24 hours after transfer, and after 6 days, popliteal LNs were collected and prepared as single-cell suspensions for flow cytometry. The cells were stained using the following primary conjugated antibodies: anti-Thy1.2–PerCP Cy5.5 (BioLegend), anti-CD4–BV650, anti-CD8–AF700, anti-CD44–BV510, anti-CD62L–PE-Cy7, CD69-FITC (all from BD Biosciences), anti-Bcl6–AF647 (BioLegend), anti–PD-1–BV711 (BioLegend), and anti-CXCR5–BV421 (BioLegend).
Anti cd62l pe cy7
Manufactured by BD
Anti-CD62L PE-Cy7 is a fluorescently labeled antibody that specifically binds to the CD62L cell surface marker. CD62L, also known as L-selectin, is a cell adhesion molecule involved in the homing of lymphocytes to peripheral lymph nodes.
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Lab products found in correlation
Anti cd44 bv510, by BD (2 mentions)
Cd69 fitc, by BD (1 mentions)
Anti cd4 bv650, by BioLegend (1 mentions)
Anti cd8 af700, by BD (1 mentions)
Anti pd 1 bv711, by BioLegend (1 mentions)
Anti cxcr5 bv421, by BioLegend (1 mentions)
Pan t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Anti cd8 po, by BioLegend (1 mentions)
Anti btla pe, by Thermo Fisher Scientific (1 mentions)
Anti 2b4 apc, by Thermo Fisher Scientific (1 mentions)
Anti pd 1 apc cy7, by Thermo Fisher Scientific (1 mentions)
Anti cd4 pb, by BD (1 mentions)
Anti cd44 percp, by BioLegend (1 mentions)
Anti cd25 fitc, by BioLegend (1 mentions)
Anti cd69 pe, by BioLegend (1 mentions)
Anti cd3 alexa 700, by BD (1 mentions)
Accucheck counting beads, by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Fc block, by Thermo Fisher Scientific (1 mentions)
Percp cy5, by BD (1 mentions)
5 protocols using anti cd62l pe cy7
1
Tracking Antigen-Specific T Cell Responses
2
Phenotypic Characterization of Splenic T Cells in Sepsis
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Mice were sacrificed and spleens were harvested 24-hours post CLP. Splenocytes were stained with anti-CD3-Alexa 700 (BD), anti-CD4-PB (BD), anti-CD8-PO and anti-CD44-PerCP (Biolegend). Cells were also surface stained with anti-CD25-FITC (Biolegend), anti-CD69-PE (Biolegend), anti-CD62L-PE Cy7 (BD), and anti-BTLA-PE, anti-2B4-APC, anti-PD-1-APC-Cy7, anti-LAG-3-FITC (all from eBioscience) for phenotypic analysis. An LSR II flow cytometer (BD Biosciences) was used to run all samples. Accucheck Counting Beads (Thermo Fisher Scientific) were added during staining to calculate the absolute number of T cells per spleen. Flow data were analyzed using FlowJo software (FlowJo, LLC) and SPADE (Cytobank.org ) (see below).
3
Multiparametric Flow Cytometry Analysis
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Cells were isolated from BAL, lungs, spleen, and draining lymph nodes and analyzed by flow cytometry as previously described55 (link). Cells were stained with the following antibodies at the concentration of 1:200: CD16/32 (i.e., Fc block, eBioscience), Fixable Viability Dye eFluor 506, anti-Ly6G PE-Cy7 or eFluor 450, anti-CD117 FITC, SiglecF PE, CD11b PE-Texas Red, anti-CD11c APC, anti-MHC II AF700, anti-CD49b PerCP eF710, anti-FcεRIα PECy7, anti-F4/80 APC/Cy7, anti-IL17A PerCP-Cy5.5 (Ebioscience), anti-TCRβ APC-CY7 (Biolegend), anti-CD4 Alexa Fluor 700 or eFluor 450, anti-CD8α PE-Texas Red or PerCP-Cy5.5, anti-TCRδ APC, anti-NK1.1 Allophycocyanin, anti-CD44 V500, anti-CD62L PE-Cy7, anti-CD11b Alexa Fluor 647 (BD Pharmingen), anti-B220 Alexa Fluor 700, PE-PBS57 loaded CD1d tetramer was from the National Institute of Allergy and Infectious Diseases Tetramer Facility. Purified anti-CD3 and CD28 antibodies were from BD Biosciences. In some cases, cells were stimulated with PMA and Ionomycin followed by an analysis of intracellular cytokine as previously described56 (link). Cells were analyzed using a BD FACS Aria II flow cytometer and analyzed with FlowJo software.
4
Murine Immune Cell Characterization
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HSF was provided by Hangzhou KSBIO Science and Technology (BinJiang, Zhejiang, China). Quality control was performed following published methods (Fu et al., 2011 (link)).
The anti-mouse antibodies for fluorescence-activated cell sorting (FACS) were as follows: anti-CD45 FITC, anti-CD8 APC-H7, anti-Eomes PE-Cy7, anti-T-bet BV421, anti-PD-1 BV605, anti-CD4 PE, anti-CD44 BV510, and anti-CD62L PE-Cy7 from BD Biosciences.
The anti-mouse antibodies for fluorescence-activated cell sorting (FACS) were as follows: anti-CD45 FITC, anti-CD8 APC-H7, anti-Eomes PE-Cy7, anti-T-bet BV421, anti-PD-1 BV605, anti-CD4 PE, anti-CD44 BV510, and anti-CD62L PE-Cy7 from BD Biosciences.
5
Phenotypic Analysis of T Cell Subsets
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Draining lymph node (dLN) and spleen cells were harvested and stained with anti-CD4-FITC, CD8-FITC, CD44-V450, CD62L-PE-Cy7, FoxP3-APC, EOMES-PE-Cy7, Bcl-6-PE, and anti-Thy1.1 (CD90.1)-PE Abs (eBioscience or BD Biosciences). To determine intracellular expression of FoxP3, EOMES, and Bcl-6, cells were fixed and permeated according to the protocol of Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent Kit (eBioscience). Then, cells were stained with anti-FoxP3, EOMES, or Bcl-6 Abs and finally analyzed using FACSAria III (BD Biosciences). To purify CD8+ TCM cells, cells were stained with anti-CD8-FITC, CD44-V450, and anti-CD62L-PE-Cy7 Abs and CD8+CD44highCD62Lhigh T cells were sorted out via FACSAria III (BD Biosciences). To purify CD3+ T cells, splenocytes were stained with anti-CD3-PE Ab and CD3+ cells then were sorted out.
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