Isolated cells from the liver were incubated with Fc block followed by incubation with fluorophore conjugated antibodies on ice in FACS buffer for the following panels: T cell panel: CD8a (PE-Cy7, 1:200, BD Biosciences), TCRβ (APC-Cy7, 1:200, BD Biosciences), CD44 (A700, 1:200, BD Biosciences), and CD62L (APC, 1:200, BD Biosciences). T cell subsets panel: CD8a (PE-Cy7, 1:200, BD Biosciences), TCRβ (APC-Cy7, 1:200, BD Biosciences), CD44 (A700, 1:200, BD Biosciences), CD62L (APC, 1:200, BD Biosciences), CXCR6 (FITC, 1:100, Biolegend), CCR7 (PerCP-Cy5.5, 1:100, Biolegend), and Ncf2 Tetramer (PE, 1:100, NIH Tetramer Core Facility). NK T cell panel: NK1.1 (PE, 1:100, BD Biosciences), TCRβ (APC-Cy7, 1:200, BD Biosciences), CD8a (PE-Cy7, 1:200, BD Biosciences), and CD4 (AF488, 1:200, BD Biosciences). Macrophage Panel: F4/80 (A700, 1:200, Biolegend), CD64 (PE, 1:200, BD Biosciences), CD11b (FITC, 1:200, BD Biosciences), Ly6c (PerCP-Cy5.5, 1:200, BD Biosciences), H2Kb (APC-eFluor780, 1:200, ThermoFisher), and H2Db (PE-Cy7, 1:200, BD Biosciences). After incubation samples were washed twice with FACS buffer. Flow data was acquired on a Becton Dickinson LSRII machine in the NCSU flow Cytometry Core. All data was analyzed using FlowJo software v10.8. Flow gating strategies are provided (Supplementary Figures 1A, B
Cd62l
Manufactured by BD
Sourced in United States, Germany
CD62L is a cell surface glycoprotein that functions as a leukocyte adhesion molecule. It plays a role in the initial attachment and rolling of leukocytes on endothelial cells during the inflammatory response.
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Annexin 5, by BD (6 mentions)
Lag 3, by BD (6 mentions)
F4 80, by BD (6 mentions)
Hla dr, by BD (6 mentions)
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Il 10, by BD (5 mentions)
7 aad, by BD (4 mentions)
Cellquest software, by BD (4 mentions)
Tcrγδ, by BD (4 mentions)
Tnf α, by BD (4 mentions)
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98 protocols using cd62l
1
Comprehensive Immunophenotyping of Liver Cells
2
Multiparametric Flow Cytometry of Immune Cells
3
Multiparametric Flow Cytometry Analysis
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All of the cells were washed and suspended in FACS
buffer that contained PBS with 0.1% sodium azide and
0.4% human serum albumin. The cells were stained with
fluorescein isothiocyanate (FITC)- and phycoerythrin
(PE)-conjugated mAbs. We used CD19, CD123, and
FRβ (Becton Dickinson, Mountain View, CA, US) to
stain the AML blasts. CD3, CD4, CD8, CD56, CD45RA,
CD45RO, CD62L, CD27, CCR7, and PD-1 (Becton
Dickinson, Mountain View, CA, US) were used to stain
the T lymphocytes. PD-1 was the exhaustion marker of
the T cells. In order to detect CAR expression, the cells
were incubated at 4o
C for 20 minutes with biotin-labelled
polyclonal goat anti-mouse F(ab)2 antibodies (Santa Cruz
Biotechnology Dallas, Texas, USA) and then washed
twice with FACS buffer. Apoptosis was measured using
Annexin V and 7AAD staining (Becton Dickinson, US).
Cells were analysed by FACSCalibur (Becton Dickinson,
US) equipped with a triple fluorescence signal filter.
buffer that contained PBS with 0.1% sodium azide and
0.4% human serum albumin. The cells were stained with
fluorescein isothiocyanate (FITC)- and phycoerythrin
(PE)-conjugated mAbs. We used CD19, CD123, and
FRβ (Becton Dickinson, Mountain View, CA, US) to
stain the AML blasts. CD3, CD4, CD8, CD56, CD45RA,
CD45RO, CD62L, CD27, CCR7, and PD-1 (Becton
Dickinson, Mountain View, CA, US) were used to stain
the T lymphocytes. PD-1 was the exhaustion marker of
the T cells. In order to detect CAR expression, the cells
were incubated at 4o
C for 20 minutes with biotin-labelled
polyclonal goat anti-mouse F(ab)2 antibodies (Santa Cruz
Biotechnology Dallas, Texas, USA) and then washed
twice with FACS buffer. Apoptosis was measured using
Annexin V and 7AAD staining (Becton Dickinson, US).
Cells were analysed by FACSCalibur (Becton Dickinson,
US) equipped with a triple fluorescence signal filter.
4
Characterization of GD2.CAR T Cells
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Approximately 500,000 cells were washed once with FACS buffer [PBS (Sigma, P3813) containing 1% fetal bovine serum], pelleted, and antibodies were added in appropriate amounts. After incubation for 20 min at 4°C in the dark, the cells were washed with FACS buffer. The GD2.CAR was detected with the 1A7 idiotypic antibody (19 (link)) followed by PE-conjugated rat anti-mouse IgG. The cells were stained with CD25, CD3, CD4, CD8, CD45RA, CD45RO, CCR7, CD62L, TCR γ/δ, CD56, CD28, PD-1, LAG-3, and CD95 monoclonal antibodies (Becton Dickinson, Franklin Lakes, NJ) TCR α/β (Biolegend, #306718) for phenotyping, and with Pro5® CLG Pentamer (ProImmune Ltd., Oxford, UK) for EBV A*02:01 LMP-2 (426–434) specific T cells. To stain for apoptosis, 100,000 cells were removed from culture, resuspended in 1X binding buffer (Becton Dickinson BD) with 5 μL of annexin V (Becton Dickinson BD,) and 4 μL of 7-AAD (Becton Dickinson BD,), and incubated at room temperature for 15 min. The cells were then immediately assessed after incubation without a washing step.
5
Multicolor Flow Cytometry Immunophenotyping
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Cell staining was performed as previously described34 (link). Cells were washed in cold PBS with 2% FCS and 0.1% sodium azide and following stained with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-conjugated anti-human monoclonal antibodies which were including anti-CD3/CD16 + CD56 (APC/FITC), NKp46, CD69, CD62L, CD158b, and CD158e (PE) from Becton-Dickinson for flow cytometric analysis. The fluorescent staining was analyzed on a Canto II (BD Biosciences) flow cytometer. Electronic gates were set to enable analysis of the fluorescence of the viable cell population according to FSC/SSC histograms following anti-CD3/ CD16 + CD56 stimulation. The percentage of cells stained with each monoclonal antibody was determined by comparing each histogram with one from control cells stained with FITC- or PE- labeled isotype control monoclonal antibodies.
6
Characterizing T-cell Functionality by Flow Cytometry
7
Ex Vivo Expansion of Antigen-Specific CD8+ T Cells
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Na€ ve CD8 T cells were isolated from allogeneic donor PBMCs via Negative Magnetic Selection (StemCell Technologies) and assessed for purity via staining for CD8, CD45RO, CD57, CCRL, and CD62L (Becton Dickinson). Isolated T cells were cocultured with DCs as described previously with the addition of 2.5 mg/mL peptide at the start of coculture (30) . T cells were expanded for 7 days, with media and cytokine refreshment on days 3 and 5, prior to evaluation for functional response. For further expansion of antigen-specific T cells, secondary stimulations were set up with a T2 cell line. T2 cells were irradiated at 100 Gy and pulsed with 2.5 mg/mL peptide, and were then cocultured with T cells at a ratio of 1:4 T-to-T2 cells for an additional 7 days in the presence of 5 ng/mL IL7 and 5 ng/mL each IL15 and IL2.
8
Multiparametric Flow Cytometry of Immune Cells
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We performed flow cytometry analysis using the following Abs: CD45, CD56, CD8, CD4, CD3, CD45RA, CD45RO, CD62L, 7AAD and Annexin V (all from Becton Dickinson, San Jose, CA) and CCR7 (from E&D) conjugated with FITC, PE, PerCP or APC fluorochromes. Expression of GD2 and CSPG4 antigens on tumor cell lines was assesed with anti-GD2 (clone 14.g2a, BD) and anti-CSPG4 (Clone 1E6.4, Miltenyi-Biotech), respectively. The expression of GD2-specific CAR was detected using a specific anti-idiotype antibody (1A7). Samples were analyzed with a BD FACScalibur system equipped with the filter set for quadruple fluorescence signals and the CellQuest software (BD Biosciences). For each sample we analyzed a minimum of 10,000 events.
9
Multiparametric flow cytometry analysis
10
Multiparametric Flow Cytometry Analysis
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Cell suspensions were incubated with 24G2 hybridoma supernatant (to block Fc receptors) and stained using immunofluorescence-labeled antibodies against the following antigens: B220/CD45R, CD3, CD19, CD11b, Ly6G, CD21, CD5, CD44, CD86, CD38, CD138, CD24, CD172a (SIRPα), CD69, CD43, CD40, CD45.2, CD93, IgM, TLR7 and CD62L from BD Biosciences, IA/IE (MHC class II), GL7, CD23, Tbet, CD4 and CD8 from eBioscience and CD11c and SiglecH from Biolegend. For intracellular TLR7 staining, cell suspensions were stained for the indicated extracellular antigens, before fixation with Cytofix (BD Biosciences). Cells were then permeabilized with saponin 0.1% and stained for TLR7. Intracellular T-bet staining was performed using the FoxP3 Fixation/Permeabilization buffer (eBioscience).
Flow cytometry was conducted using an LSR2 (BD Biosciences) and data were analyzed with FlowJo (Tree Star). The full gating strategy that used for flow cytometry analysis is presented in Supporting Information Fig. 6. For splenocyte proliferation experiments, erythrocyte-depleted splenocytes were labeled with CellTrace Violet (Life technologies) according to manufacturer's recommendations.
After 4 days of stimulation, cells were harvested and stained with antibodies for analysis by flow cytometry.
Flow cytometry was conducted using an LSR2 (BD Biosciences) and data were analyzed with FlowJo (Tree Star). The full gating strategy that used for flow cytometry analysis is presented in Supporting Information Fig. 6. For splenocyte proliferation experiments, erythrocyte-depleted splenocytes were labeled with CellTrace Violet (Life technologies) according to manufacturer's recommendations.
After 4 days of stimulation, cells were harvested and stained with antibodies for analysis by flow cytometry.
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