Target K562-EV/CD1a cells were fluorescently labelled with CellTraceViolet (Invitrogen) prior to the infection. CD1a-restricted T cell lines/clones (1-5x105) were added to non-infected/infected target K562-EV/CD1a cells (2x105) in the presence of IL-12 (1 ng/mL; BioLegend) and IL-18 (1 ng/mL; BioLegend) and IL-2 (25 U/mL; BioLegend). Supernatant was collected after 24 hr co-culture for cytokine analysis. Cell death was assessed by flow cytometry after 48 hrs co-culture. Briefly, the wells were harvested, and to stain for dead and apoptotic cells, Zombie Fixable Viability dyes (1:1000; BioLegend) and Annexin V-APC (BioLegend) were added. To allow quantitative analysis of the target cell populations, 2x105 CFSE-labelled K562 cells (as reference cells) were added. This was done just prior to the FACS analysis to avoid the interaction between the target, reference, and T cells. Data were acquired using LSRFortessa X-50 flow cytometer (BD Biosciences) and further analyzed with FlowJo (FlowJo LLC) software. The percentage of induced killing was then calculated with the following equation by comparing the frequency of live target and reference populations: % cytotoxicity = 100-((% live target cells /% live reference cells)/(% live cells of untreated K562-EV/% live reference cells) x 100).
Zombie fixable viability dye
Manufactured by BioLegend
The Zombie fixable viability dye is a fluorescent stain used to detect dead cells in flow cytometry applications. It binds to cellular proteins, providing a reliable method for distinguishing between live and dead cells in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (6 mentions)
Anti cd16 32 2.4g2, by BioXCell (4 mentions)
Automacs running buffer, by Miltenyi Biotec (3 mentions)
Annexin 5 apc, by BioLegend (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm plus kit, by BD (2 mentions)
Anti biotin pe antibody, by Miltenyi Biotec (2 mentions)
Fortessa, by BD (2 mentions)
Anti mouse cd16 32, by BioXCell (2 mentions)
Foxp3 tf staining buffer set, by Thermo Fisher Scientific (2 mentions)
Macsquant, by Miltenyi Biotec (2 mentions)
Trustain fcx anti cd16 cd32, by BioLegend (2 mentions)
Fitc conjugated anti dectin 1 antibody, by Bio-Rad (2 mentions)
Facsymphony, by BD (2 mentions)
Il 18, by BioLegend (1 mentions)
Lsrfortessa x 50 flow cytometer, by BD (1 mentions)
Il 12, by BioLegend (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by BioLegend (1 mentions)
21 protocols using zombie fixable viability dye
1
CD1a-Restricted T Cell Cytotoxicity Assay
2
Multiparametric Flow Cytometry of Tumor Cells
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Single-cell suspensions from tumors were generated using the mouse tumor dissociation kit with the gentleMACS Octo Dissociator according to the manufacturer’s protocol (Miltenyi Biotec). Cells were labeled with fluorochrome-conjugated antibodies for 30 min at 4°C. Live/Dead cell discrimination was performed by addition of Zombie Fixable Viability™ Dyes (BioLegend). For intracellular staining, cells were fixed and permeabilized using a Cytofix/Cytoperm Kit (BD Biosciences) according to the manufacturer’s instructions as preparation for intracellular staining using fluorescently labeled antibodies in BD Perm/Wash™ buffer (BD Biosciences). Antibody staining was performed in the presence of purified 2.4G2 antibody (Fc block). Apoptotic cells were detected using the FITC Annexin V Apoptosis Detection Kit with PI (Biolegend) according to the manufacturer’s instructions. Antibodies for flow cytometry were purchased from either BD Biosciences, Invitrogen, or BioLegend. Flow cytometry was performed using BD LSRFortessa™ or BD FACSLyric™ instruments (BD Biosciences). A list of flow cytometry antibodies is available in the Supplementary Information. Data were analyzed using FlowJo software (Tree Star, Inc., Version 10).
3
Flow Cytometry Immunophenotyping Protocol
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Flow cytometry measurements were performed using a LSRII or LSR Fortessa instrument (BD Biosciences). Data were analyzed with FlowJo software (BD Biosciences). Fluorescently conjugated antibodies were purchased from Biolegend, eBioscience/Thermo Fisher Scientific and BD Biosciences. Intracellular staining was performed using a Cytofix/Cytoperm Plus Kit (BD Biosciences) or a FoxP3/Transcription Factor Staining Buffer Set (eBioscience/Thermo Fisher Scientific). Surface staining was performed in PBS +2% FBS. For exclusion of dead cells, Zombie Fixable Viability Dyes were used (Biolegend).
4
Quantifying CD1a-Mediated Cell Death
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Anti-CD1a antibodies (5 µg/ml) or commercially available comparator NA1/34 were incubated with 1 × 105 CD1a-expressing K562 or EV control K562 for 24–48 h and cell death assessed by flow cytometry. Briefly, some wells were harvested after 24 h culture, and to stain for dead and apoptotic cells, Zombie Fixable Viability dyes (1:1000; BioLegend) and Annexin V-APC (BioLegend) were added. To measure direct antibody-induced cell death after 48 h, K562 were fluorescently labelled with CellTraceViolet prior to incubation with anti-CD1a antibodies. To allow quantitative analysis of the surviving target cell populations, 2 × 105 CFSE-labelled K562 (Reference) were added. This was done just prior to the analysis to avoid the interaction between the target, reference, and T cells. The percentage of induced killing was then calculated with the following equation by comparing the frequency of live target and reference populations: % cytotoxicity = 100−((% live CD1a-antibody-treated target cells /% live reference cells)/(% live isotype-antibody-treated target cells/% live reference cells) × 100).
5
Popliteal Lymph Node Immune Cell Isolation
6
FcγR-Mediated BMDC Maturation and Cytokines
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To assess FcγR dependent BMDC maturation, ova ICs were added directly to culture wells on day 6–7 of culture; ova or rabbit anti-ova alone were used as controls. 24 hours later, BMDCs were harvested, washed, blocked with a non-fluorescent FcγRII/III antibody (clone 2.4G2, BioLegend) and stained for cell surface expression of CD54, CD80, CD86 and MHCII I-Ab (clones YN1/1.7.4, 16-10A1, GL-1 and AF6-120.1 respectively, BioLegend). To identify live DCs, cells were also stained with anti-CD11c (clone N418, BioLegend) and a viability marker (Zombie Fixable Viability dye, BioLegend). Cells were washed with FACS buffer (5% FBS, 1 mM EDTA in PBS) and fixed using 1% paraformaldehyde (PFA, Electron Microscopy Sciences) in PBS prior to flow cytometry. Maturation marker expression was determined by gating on live CD11c+ singlets.
To determine FcγR induced cytokine production, BMDCs were harvested on day 7 of culture, counted and replated in 96 well round bottom plates at 2 × 105/well, and stimulated with ova, rabbit anti-ova, ova pre-incubated with rabbit IgG, or ova ICs for 3–24 hours, in the presence of polymyxin B (50 µg/ml, Sigma). Cell-free supernatants were removed and assessed for IL-6, TNFα, and IL-12/23p40 cytokine expression by immunoassay as per the manufacturer’s instructions (BioLegend).
To determine FcγR induced cytokine production, BMDCs were harvested on day 7 of culture, counted and replated in 96 well round bottom plates at 2 × 105/well, and stimulated with ova, rabbit anti-ova, ova pre-incubated with rabbit IgG, or ova ICs for 3–24 hours, in the presence of polymyxin B (50 µg/ml, Sigma). Cell-free supernatants were removed and assessed for IL-6, TNFα, and IL-12/23p40 cytokine expression by immunoassay as per the manufacturer’s instructions (BioLegend).
7
Comprehensive Multiparametric Flow Cytometry
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Cells were first stained with Zombie Fixable Viability dye (Biolegend) for 10 minutes at room temperature (RT). For surface staining, cells were stained with antibodies in autoMACS running buffer (Miltenyi) containing 5 ug/ml of anti-mouse CD16/32 (BioXcell) for 20 minutes at 4°C. In some experiments, TB10.44-11 and/or 32A309-318 tetramers were used with other antibodies. To measure transcription factor (TF) expression, cells were fixed and permeabilized for 30 minutes at RT, followed by staining for 30 minutes with antibodies to the different TF at RT using the Foxp3/TF staining buffer set (ThermoFisher). Samples were acquired on Aurora (Cytek) or MACSquant (Miltenyi). Flow data were analyzed using FlowJo v10.7.1, and FlowJo plugins UMAP v3.1 and PhenoGraph v3.0. For UMAP projections of WT/KO CD8 T cells (i.e., Figures 3C and S1C ), 2000 CD45iv−CD44+CD62L− CD8 T cells/mouse from 5 WT and 5 MHCII KO mice were concatenated to one FCS file before creating UMAP projections. In UMAP projections comparing two models (i.e., Figure 5D ), 2000 CD45iv−CD44+CD62L− CD8 T cells/mouse from 5 WT and 5 KO, and 6 helped and 4 helpless mice from adoptive transfer model were concatenated and created UMAP projections. K = 300 and K = 400 were used in PhenoGraph analysis in Figures 3D and 5B , respectively, and the resulting clusters were overlayed onto UMAP projections.
8
Dectin-1 Expression Analysis
9
MHC-I and TNFRI Expression Analysis
10
Single-cell Staining and Sorting Protocol
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Single-cell suspensions were washed with PBE, incubated with 1 μg ml−1 anti-CD16/32 (2.4G2, BioXCell) for 5 min at room temperature and then stained for cell surface markers at 4 °C for 20 min in PBS using the reagents listed in Supplementary Table 1 . Cells were washed with PBE and stained with Zombie fixable viability dye (BioLegend) or fixable Aqua dead cell stain kit (Invitrogen) at room temperature for 15 min, then washed with PBE and filtered through a 40 μm strainer for acquisition. For in vivo JAML staining of IELs, mice were injected i.p. with 100 μg of anti-JAML AF646 antibody 12 or 6 h prior to the end point. For single-cell transcriptomic analysis, stained cells were further incubated with DNA-barcoded anti-biotin and sample hashtag (anti-MHC-I) antibodies (BioLegend) for 20 minutes in PBE, washed three times with PBE, and bulk-sorted. For substrate detection in vivo, an anti-biotin–PE antibody (Miltenyi Biotec) was exclusively used, as described previously59 (link). Samples were acquired on FACS Symphony or Fortessa analyzers or sorted on FACSAriaII or FACSAriaIII cell sorters (BD Biosciences). Data were analyzed using FlowJo v.10.6.2 software.
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