Cells were stained with Aqua Live/Dead viability dye (Life Technologies, Carlsbad, USA), followed by extracellular antibody staining for 30 min at room temperature. Antibodies, clones, fluorochromes, and manufacturers are summarized in Table S2. Samples were analyzed with a BD LSR Fortessa flow cytometer using BD FACSDiva software (BD Bioscience, Franklin Lakes, USA). Data were analyzed using FlowJo v10.2 (FlowJo LLC, Ashland, USA). The gating strategy for phenotypic analyses of AF-CAR T cells and a representative data set are shown in Fig. S5.
Aqua live dead viability dye
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The Aqua Live/Dead viability dye is a fluorescent stain used to assess cell viability in flow cytometry applications. It is capable of distinguishing between live and dead cells by selectively staining dead cells.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (5 mentions)
Fc block, by Thermo Fisher Scientific (4 mentions)
Sorp lsr 2, by BD (3 mentions)
Cytofix cytoperm, by BD (3 mentions)
Lsr 2 flow cytometer, by BD (2 mentions)
Aria 2, by BD (2 mentions)
Fortessa flow cytometer, by BD (2 mentions)
F4 80, by Thermo Fisher Scientific (2 mentions)
Cfse proliferation dye, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Pe anti nkg2a z199, by Beckman Coulter (2 mentions)
Foxp3 transcription factor staining buffer, by Thermo Fisher Scientific (2 mentions)
Golgiplug, by BD (2 mentions)
Rhil 2, by BioLegend (2 mentions)
Rhil 7, by BioLegend (2 mentions)
Anti cd4 bv785 okt4, by BioLegend (2 mentions)
Anti cd26 pe cy7 ba5b, by BioLegend (2 mentions)
Rhil 12, by BioLegend (2 mentions)
Anti cd3 percp cy5.5 ucht1, by BioLegend (2 mentions)
Rhil 15, by BioLegend (2 mentions)
22 protocols using aqua live dead viability dye
1
Phenotypic Analysis of AF-CAR T Cells
2
Immunophenotypic Analysis of Cell Viability
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After cells were counted, and 2x106 cells per sample were stained with Aqua Live/Dead viability dye (Life Technologies) according the manufacturer’s instructions. Cells were then incubated in blocking solution containing 5% normal mouse serum, 5% normal rat serum, and 1% FcBlock (eBiosciences, San Diego, CA) in PBS and then stained with a standard panel of immunophenotyping antibodies (See S2 Table for a list of antibodies, clones, fluorochromes, manufacturers, and concentrations) for 30 minutes at room temperature. After staining, cells were washed and fixed with 0.4% paraformaldehyde in PBS. Data was acquired with a BD LSRII flow cytometer using BD FACSDiva software (BD Bioscience). Compensation was performed on the BD LSRII flow cytometer at the beginning of each experiment. Data were analyzed using Flowjo v10. Cell sorting for cytospins was performed on a BD Aria II. The collected cells were stained with a Jenner-Giemsa Stain Kit (ENG Scientific Inc, Clifton, NJ) and examined by light microscopy.
3
CD4+ T-cell Proliferation Assay
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We assessed CD4+ T-cell proliferation, as previously described (13 (link), 15 (link)). CFSE-labeled PBMCs were treated in culture medium for six days with sorted CD27+ MPs at ratios of 2000:1, 200:1, 20:1, 1:1 and 1:10 (PBMC: sorted CD27+ MPs). Lymphoproliferation was measured by flow cytometry analyses of CD4+ TLs with anti-CD4-APC-H7 and anti-CD3-PE (BD Biosciences) antibodies. Aqua Live/Dead viability dye (Thermo Fisher Scientific, MA, Waltham) was added to exclude dead cells. Lymphoproliferation was normalized between donors. For each HD tested, a proliferation index of 1 was assigned to the lymphoproliferation observed in the absence of MPs. Lymphoproliferation is expressed proportionally, as the fold-induction relative to lymphoproliferation in the absence of MPs.
4
SARS-CoV-2 RBD-Specific T Cell Response
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Mouse spleens were
harvested on D105 after vaccination, and single-cell suspensions were
prepared by processing them through a 70 μm cell strainer (BD
Biosciences). Cells were then incubated in FACS tubes at 6 ×
105 cells per tube and stimulated for 18 h with 2 μg/mL
RBD peptide mix [PepMix SARS-CoV-2 (S-RBD) Protein ID: P0DTC2 PM-WCPV-S-RBD-1,
JPT] or media alone. As a positive control, cells were stimulated
with antimouse CD3 [0.05 μg/mL] (Southern Biotech) and antimouse
CD28 antibody [0.5 μg/mL] (Southern Biotech). GolgiStop (BD
Biosciences) was added for the last 10 h of the assay. Following stimulation,
cells were washed, stained with Aqua live/dead viability dye (Thermo
Fisher) in PBS, washed two additional times, and stained with a cocktail
of monoclonal antibodies and Fc block: CD16/32, CD4 Ax700 RM4-5, CD8
APC-H7 53-6.7, and CD44 APC IM7 (all BD Bioscience). Cells were fixed
and permeabilized according to the manufacturer’s protocol
(BD Biosciences) and stained for intracellular cytokines with IFNγ
PE-Cy7 XMG1.2, TNFα BV650 MP6-XT22, and IL-4 BV786 11B11 (BD
Bioscience). Cells were washed, fixed with 2% formaldehyde, acquired
on a Cytek Aurora (Northern Lights), and analyzed using Cytobank V7.3.0.
harvested on D105 after vaccination, and single-cell suspensions were
prepared by processing them through a 70 μm cell strainer (BD
Biosciences). Cells were then incubated in FACS tubes at 6 ×
105 cells per tube and stimulated for 18 h with 2 μg/mL
RBD peptide mix [PepMix SARS-CoV-2 (S-RBD) Protein ID: P0DTC2 PM-WCPV-S-RBD-1,
JPT] or media alone. As a positive control, cells were stimulated
with antimouse CD3 [0.05 μg/mL] (Southern Biotech) and antimouse
CD28 antibody [0.5 μg/mL] (Southern Biotech). GolgiStop (BD
Biosciences) was added for the last 10 h of the assay. Following stimulation,
cells were washed, stained with Aqua live/dead viability dye (Thermo
Fisher) in PBS, washed two additional times, and stained with a cocktail
of monoclonal antibodies and Fc block: CD16/32, CD4 Ax700 RM4-5, CD8
APC-H7 53-6.7, and CD44 APC IM7 (all BD Bioscience). Cells were fixed
and permeabilized according to the manufacturer’s protocol
(BD Biosciences) and stained for intracellular cytokines with IFNγ
PE-Cy7 XMG1.2, TNFα BV650 MP6-XT22, and IL-4 BV786 11B11 (BD
Bioscience). Cells were washed, fixed with 2% formaldehyde, acquired
on a Cytek Aurora (Northern Lights), and analyzed using Cytobank V7.3.0.
5
Multicolor Flow Cytometry of Retinal and Blood Cells
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Single cell suspensions of retina or lysed blood were transferred into PBS for staining with Aqua Live/Dead viability dye (Thermo Fisher Scientific, Waltham, MA) for 30 minutes then washed. Cells were incubated in a blocking solution containing 5% normal mouse serum, 5% normal rat serum, and 1% Fc Block (eBiosciences, San Diego, CA) and subsequently stained with a combination of fluorophore-conjugated primary antibodies against CD45, Ly6C, Ly6G, CD64, CD11c, CD11b (Biolegend), F4/80 (eBiosciences), I-A/I-E (BD Biosciences, San Jose, CA) and CCR2 (R&D Systems, Minneapolis, MN). Samples were stained at room temperature for 20 minutes; then a fluorophore-conjugated streptavidin (Biolegend) was added to each sample and staining continued for an additional 5 minutes. After completion of staining, cells were washed and fixed with 0.4% paraformaldehyde in PBS. Data was acquired with BD Fortessa flow cytometer using BD FACSDiva software (BD Biosciences). Raw flow cytometry data was analyzed using FlowJo software (FlowJo LLC, Ashland, OR)
6
Cytokine-Mediated T Cell Polarization
7
Isolation and Sorting of B Cell Subsets
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Isolated PBMCs from a healthy adult volunteer were incubated with 100 µL Aqua Live/Dead viability dye (Thermo Scientific, UK) per 1 × 106 cells for 20 min at room temperature in the dark. Viability dye was prepared by diluting 1:400 in PBS. Cells were then washed with PBS, and incubated with the pre-titrated B cell phenotyping panel shown in Table S5 for 30 min at room temperature in the dark. Following incubation, cells were washed with PBS and re-suspended in warm RPMI 1640 medium (Life Technologies, UK) supplemented with 100 U/mL penicillin (Life Technologies), 100 µg/mL streptomycin (Life Technologies), 2% FCS, and 2 mM l -glutamine (Life Technologies) (R2 medium). CD27+ memory B cells were sorted into four distinct populations based on IgM, IgD, and IgG expression (IgD+ IgM+, IgD− IgM+, IgD− IgM− IgG−, and IgD− IgM− IgG+). Cells were sorted directly into 1.5 mL microcentrifuge tubes containing RPMI 1640 medium supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM l -glutamine, and 10% FCS. Sorting was carried out using a BD FACSAria III and data analyzed using FlowJo software 9.7.5 (TreeStar, Ashland, OR, USA). Antibody panels used are provided in Table S5 in Supplementary Material.
8
Eye Macrophage Immunophenotyping Protocol
9
Murine T-cell Polarization Assay
10
Immunophenotypic Analysis of Immune Cells
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After cells were counted, 2×106 cells per sample were stained with Aqua Live/Dead viability dye (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Cells were then incubated in blocking solution containing 5% normal mouse serum, 5% normal rat serum, and 1% FcBlock (eBioscience; Thermo Fisher Scientific, Inc.) in PBS at 4°C for 1 h, and then stained with a standard panel of immunophenotyping antibodies (CD11c, 1:800, cat. no. ab254183; CD80, 1:800, cat. no. ab134120; CD3, 1:1,000, cat. no. ab16669; CD8, 1:500, cat. no. ab217344 and DX5, 1:200, cat. no. ab181548; Abcam) for 30 min at room temperature. After staining, cells were washed and fixed with 0.4% paraformaldehyde in PBS at 4°C for 24 h. Data was acquired with a BD LSRII flow cytometer using BD FACSDiva software (v 8.0.3; BD Biosciences). Compensation was performed on the BD LSRII flow cytometer at the beginning of each experiment. Data were analyzed using Flowjo v10 (FlowJo, LLC). Cell sorting for cytospins was performed on a BD Aria II. The collected cells were stained with a Jenner-Giemsa Stain kit (ENG Scientific Inc.) at room temperature for 15–30 min and examined by light microscopy (magnification, ×40).
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