The following conjugated monoclonal antibodies from eBioscience/ThermoFisher were used: CD3 APC-efluor780 (17A2), CD4 efluor450 (RM4-5), CD45.2 APC (104), CD62L PE-Cy7 (MEL-14), CD69 PE (H1.2F3), Foxp3 APC (FJK-16s), IL-17A PE-Cy7 (eBio17B7), IFN-gamma efluor660 (XMG1.2), ROR-gamma-t PE (B2D), and T-bet efluor660 (eBio4B10). Dead cells were excluded by LIVE/DEAD® Fixable Dead Cell Stain Kit (Life Technologies). For intracellular cytokine staining, cells were stimulated with Phorbol 12-myristate 13-acetate (0.1 μg/ml/1; Sigma-Aldrich) and ionomycin (1 μg/ml/1; Sigma-Aldrich) for 4h, the last 2h in the presence of Brefeldin A (5 μg/ml), stained for surface markers, fixed using Foxp3/Transcription Factor Fixation/Permeabilization Kit (Affymetrix/eBioscience) according to manufacturer’s instruction, and stained with the respective antibodies against cytokines or transcription factors diluted in PBS containing 0.25% BSA and 0.5% of Saponin. The acquisition was performed on an LSR II flow cytometer (Becton Dickinson), and data were analyzed with FlowJo software (Tree Star, Inc.). Single stains were used for compensation and fluorescence minus one (FMO) controls for gating. For the MT-CO1 (Abcam, EPR19628), staining cells were fixed with ice-cold methanol.
Cd62l pe cy7
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The CD62L-PE-Cy7 is a fluorescently labeled antibody that binds to the CD62L (L-selectin) antigen on the surface of cells. It is commonly used in flow cytometry applications for the identification and analysis of cell populations expressing the CD62L marker.
Automatically generated - may contain errors
Lab products found in correlation
Foxp3 apc, by Thermo Fisher Scientific (3 mentions)
Cd103 apc, by Thermo Fisher Scientific (3 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Cd45.1 pe, by Thermo Fisher Scientific (2 mentions)
F4 80 percp cy5, by BioLegend (2 mentions)
Cd11b bv650, by BioLegend (2 mentions)
Pd l1 pe, by BioLegend (2 mentions)
Cd4 bv421, by BioLegend (2 mentions)
Pd1 bv605, by BD (2 mentions)
Cd90.1 fitc, by Thermo Fisher Scientific (2 mentions)
Cd45.2 alexa700, by Thermo Fisher Scientific (2 mentions)
Cd8α percp cy5, by Thermo Fisher Scientific (2 mentions)
Cd24 apc cy7, by Thermo Fisher Scientific (2 mentions)
Cd44 apcy7, by Thermo Fisher Scientific (2 mentions)
Cd90.2 alexa 700, by BioLegend (2 mentions)
Mhcii bv421, by BioLegend (2 mentions)
Cd8α pe texas red, by Thermo Fisher Scientific (2 mentions)
Ly6c bv711, by BioLegend (2 mentions)
Cd3e pe, by BD (2 mentions)
Cd45 bv786, by BD (2 mentions)
12 protocols using cd62l pe cy7
1
Comprehensive T Cell Immunophenotyping
2
Flow Cytometry Analysis of Stimulated T Cells
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Splenocytes were stimulated with a mix of peptide pools P1+P8 for tHIVconsvX and PI+PIII for CH505 at 2 μg/mL per peptide or a tissue culture medium with 1% DMSO as a negative control. The cells were washed with PBS (pH 7.2) plus 0.5% BSA and 2 mM EDTA, labeled with 10 μL of IFN-γ-catch reagent for 5 min at 4°C followed by the addition of 1 mL of warm media and incubated at 37°C for 45 min on a tube rotator (VWR). Immediately following incubation, cells were placed on ice for 5 min, washed, and stained at 4°C for 10 min with 100 μL of a mastermix containing anti-IFN-γ phycoerythrin (PE) mAb (Miltenyi Biotec) with LIVE/DEAD fixable aqua stain (Molecular Probes, Invitrogen) and the anti-membrane marker mAbs CD3 allophycocyanin (APC), CD4 fluorescein isothiocyanate (FITC), CD8a eFluor 450, CD44 Alexa Fluor 700, and CD62-L PE-Cy7 (all from Thermo Fisher Scientific). Cells were washed and fixed with 1% paraformaldehyde in PBS prior to running on an LSRII flow cytometer (Becton Dickinson). The frequencies of the subtypes in CD8+ and CD4+ T cells represent the differences in stimulated and unstimulated immune cells. All data are shown after subtracting the background values.
3
Tetramer-based Quantification of Antigen-specific T Cells
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STING KO mice and wild-type littermates were immunized as described above. Spleens were removed 7 days after primary immunization. Isolated splenocytes were directly used for staining with B8- or OVA-reactive tetramers. Briefly, splenocytes were washed with PBS and dead cells excluded by viability dye staining (eBioscienceTM Fixable Viability Dye eFluorTM 506). Thereafter, cells were washed twice with staining buffer (1% BSA, 0.02% NaN3 in PBS) and Fc-receptors blocked using CD16/32 antibodies (Fc-BlockTM, BD Biosciences). After washing, cells were incubated with B8- and OVA-reactive tetramers for 15 min and incubated in the dark on ice. Anti-CD8-PB, -CD127-APC, and -CD62L-PE/Cy7 (all eBioscience) antibodies were added to the tetramer-stained cells and incubated for additional 20 min in the dark on ice. Samples were analyzed by flow cytometry using BD FACS Canto II (BD Biosciences, Heidelberg, Germany).
4
Immune Profiling of Tumor-Bearing Mice After MVA-TWIST/TRICOM Vaccination
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Expression of B7-1, ICAM-1, and LFA-3 was determined by flow cytometry. Treated MC38 cells were stained with FITC-labeled antibodies to CD80 (B7-1), CD54 (ICAM-1) and CD48 (LFA-3) (BD Biosciences, San Jose, CA). Cells were incubated with antibodies for 30 min at 4°C. Samples were acquired on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The effect of MVA-TWIST/TRICOM on splenic immune cell populations was examined in tumor bearing and non-tumor bearing BALB/c mice 17 days after receiving two vaccinations with MVA-TWIST/TRICOM or being left untreated (n = 5/group). Vaccination of tumor bearing mice began 4 days post-implantation of 5 × 104 4T1 mammary tumor cells. Spleens were prepared and stained as described previously [60 (link)], using the following antibodies: CD3e-V500, t-APC, CD8a-Pacific Blue, CD25-FITC, CD44- PerCP-Cy5.5 CD11b-V500, Gr-1-APC, CD11c-PerCP-Cy5.5, CD40-FITC (BD Biosciences); CD62L-PE-Cy7, FoxP3-PE, MHC II-efluor450 (eBioscience, San Diego, CA); and CD49b-PE-Cy7 (Biolegend, San Diego, CA). Tetramer staining (Beckman Coulter, Pasadena, CA) was performed on splenocytes from non-tumor bearing mice following 7 days of in vitro stimulation with 1.0 μg/mL Twist peptide (BALB/c - LYQVLQSDEL). All samples were acquired on a BD Verse flow cytometer. All marker expression was determined using FlowJo software (TreeStar, Inc., Ashland, OR).
5
Multiparametric Flow Cytometry Analysis
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Flow-cytometry antibodies included CD3e-PE, PD1-BV605, CD45-BV786 (BD-Biosciences), CD90.1-FITC, CD62L-PECy7, CD45.1-APC, CD45.1-PE (eBioscience), CD45.2-Alexa700, CD-8α-Percp-Cy5.5, CD4-FITC, CD103-APC, CD24-APCcy7 (Invitrogen), CD44-APCy7, CD4-BV421, F4/80-PercpCy5.5, CD39-PECy7, PDL1-PE, CD90.2-Alexa 700, MHCII-BV421, CD11b-BV650, Ly6C-BV711 (Biolegend), CD8α-PE-Texas-red (Life Technologies, Carlsbad, CA, USA). Blocking PD-1 antibody (clone RMP1-14, BioXCell, Branford, CT, USA) was administered systemically by intraperitoneal injections of 250 μg [41 (link)]. Blocking anti-CD40L (clone MR1, BioXCell) was administered systemically by intraperitoneal injections of 250 μg [19 (link)].
6
Safety Vein Cannulation and Sample Preparation
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Safety-Multifly vein cannulas (21 gauge (G)) and 2.7 ml S-Monovettes were purchased by Sarstedt (Nümbrecht, Germany), VenflonPro Safety vein catheters (18G), Vacutainer Eclipse vaccum cannulas (22G) and the corresponding vacutainer sample vessels were purchased from Becton Dickinson GmbH (Heidelberg, Germany). B. Braun Omnifix Solo Luer syringes 10 ml were obtained by B. Braun Melsungen AG (Melsungen, Germany). Pancoll human, Density: 1.077 g/ml, was purchased by PAN-Biotech GmbH (Aidenbach, Germany) and sodium citrate 3.13% was purchased by EIFELFANGO Chem. Pharm. Werk GmbH & Co. KG (Bad Neuenahr-Ahrweiler, Germany). Calcium free RPMI 1,640 Medium was purchased from United States Biological (Salem, MA, USA). Dimethylsulfoxid (DMSO), Dihydrorhodamine 123 (DHR) and N-Formylmethionine-leucyl-phenylalanine (fMLP) were purchased from Merck KGaA (Darmstadt, Germany). Antibodies for flow cytometry analysis (CD66b—FITC, CD11b—APC-Cy7, CD45—VioBlue, CD16—VioGreen and CD62L—PE-Cy7) were purchased from eBioscience (Frankfurt am Main, Germany).
7
Multiparameter Flow Cytometry Panel
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Example 3
Antibodies for cell surface staining were obtained from BD Biosciences (anti-human CD8-PE-Cy7, CD8-V500, CD45RA-APC, CD62L-PECy7), eBioscience (anti-human CD45RO-eFluor650), Immudex and Proimmune (HLA-A201-HBs183-91-PE dextramer or pentamer), and R&D Systems (human LTβ receptor-Fc chimera). Antibodies for intracellular cytokine staining were obtained from BD Biosciences (anti-human IFN-γ-APC, TNF-α-Alexa488, IL-2-PE, Granzyme-APC) and Diaclone (anti-human perforin-FITC). Intracellular cytokine staining was performed by fixing and permeabilizing cells with cytofix/cytoperm (BD Biosciences). Flow cytometry was performed using a FACS Canto flow cytometer or LSRII (BD Biosciences) and data was analyzed with FACS Diva program (BD Biosciences).
8
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.
9
Multicolor FACS Analysis of Immune Cells
10
Comprehensive Murine and Human Immune Profiling
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Flow cytometry antibodies for murine samples included CD3e-PE, PD1-BV605, CD45-BV786 (BDbiosciences), CD90.1-FITC, CD62L-PECy7, CD45.1-PE (ebioscience), CD45.2-Alexa700, CD-8α-Percp Cy5.5, CD4-FITC, CD103-APC, CD24-APCcy7 (Invitrogen), CD44-APCy7, CD4-BV421, F4/80-PercpCy5.5, CD39-PECy7, PDL1-PE, CD90.2-Alexa 700, MHCII-BV421, CD11b-BV650, Ly6C-BV711 (Biolegend), CD8α-PE-Texas red (life technologies), CD278 (ICOS)-PE (Biolegend). Flow cytometry antibodies for human samples included CD45-BV510, CD3-Alexa700, CD4-BV785, CD8-BV711, CTLA-4-PE/Dazzle 594, 4-1BB-PE (Biolegend), CD39-BV650, PD-1-PE-Cy7 and Ki-67 Alexa 488 (BD Biosciences), CD103-APC and FOXP3-Alexa 700 (eBioscience).
Mouse antibodies for the explant assays included anti-PD1 (RMP1-14) and anti-CTLA-4 (9D9) by BioXCell and OX40 (OX86) kindly provided by Dr. Andrew Weinberg (EACRI). Human antibodies for the explant assays included anti-PD1 and anti-CTLA4 purchased from Invitrogen and anti-OX40 kindly provided by Dr. Andrew Weinberg (EACRI).
Mouse antibodies for the explant assays included anti-PD1 (RMP1-14) and anti-CTLA-4 (9D9) by BioXCell and OX40 (OX86) kindly provided by Dr. Andrew Weinberg (EACRI). Human antibodies for the explant assays included anti-PD1 and anti-CTLA4 purchased from Invitrogen and anti-OX40 kindly provided by Dr. Andrew Weinberg (EACRI).
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