For human studies, thawed PBMCs were stained with Zombie UV viability dye (1:500; Biolegend) at room temperature for 20 minutes, washed and then stained with primary antibodies on ice for 30 minutes before being acquired on a BD Fortessa X-20 (BD Biosciences). Human Basophils were defined as live CD123+ FcεRIα+ cells that lacked expression of c-Kit and lineage (Lin) markers CD3, CD4, CD19, CD14, CD34, and CD56. arker CD203c was included in the primary antibodies to reveal the physiological state of human basophils. For animal studies, 50–100 µL of blood was collected into EDTA coated tubes, followed by RBC lysis using RBC lysis buffer (Sigma-Aldrich) at room temperature for 5 minutes twice and washed by PBS once. All cells were stained with Zombie UV viability dye (1:500; Biolegend) for viability at room temperature for 20 minutes, followed by primary antibodies on ice for 30 minutes prior to data acquisition on a BD Fortessa X-20 (BD Biosciences). Basophils were defined as live CD49b+ FcεRIα/IgE+ cells that were negative for expression of c-Kit and Lin markers CD3e, CD5, CD11c, CD19, and NK1.1. The mouse basophil canonical activation marker CD200R was also stained. All flow cytometry data were analyzed with Flowjo v10 software (Tree Star).
Zombie uv viability dye
Manufactured by BioLegend
Sourced in United States
Zombie UV viability dye is a fluorescent dye used to stain and identify dead or dying cells. It specifically binds to nucleic acids in cells with compromised cell membranes, allowing for the detection of non-viable cells through fluorescence microscopy or flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Cell staining buffer, by BioLegend (2 mentions)
Fortessa x20, by BD (2 mentions)
Facs symphony a5 instrument, by BD (1 mentions)
Anti cd14 v500, by BD (1 mentions)
40 μm cell strainer, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Fortessa cell analyzer, by BD (1 mentions)
Cellstripper, by Corning (1 mentions)
Anti cd16 32 antibody, by BioLegend (1 mentions)
Moflo astrios eq cell sorter, by Beckman Coulter (1 mentions)
Human trustain fcx, by BioLegend (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Fix perm buffer, by Thermo Fisher Scientific (1 mentions)
Facsymphony flow cytometer, by BD (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
True nuclear transcription factor buffer set, by BioLegend (1 mentions)
Primeflow rna assay kit, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
16 protocols using zombie uv viability dye
1
Identification of Human and Mouse Basophils
2
Co-culture of Liver Tissue and CD14+ Cells
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Tumor and non-tumorous liver tissue from HCC patients was manually cut into small tissue fragments of 1-2 mm3. After processing, tissue fragments were placed in ultra-low adherence Nunclon™ Sphera™ 96-Well U-Shaped-Bottom Microplates in 100 μL of complete medium (RPMI-1640, 10% FBS, 1X GlutaMax, 1X Penicillin/Streptomycin, 1X Hepes, 1X non-essential amino acids, 1X Sodium Pyruvate, 0.01% β-mercaptoethanol). CD14+ cells were isolated from peripheral blood from the same patients. CD14+ cells were stained using the CellTrace Far Red (CTFR) Cell Proliferation kit (Biolegend) according to the manufacturer's protocol and 50,000 cells CTFR+ CD14+ cells were added to the wells containing tissue fragments. CTFR+ CD14+ cells were cultured alone as controls. After 3-7 days of co-culture cells and tissues were recovered, disaggregated mechanically, and filtered through 40 μm cell strainers (BD). Cell suspensions were stained with Zombie UV viability dye (Biolegend), anti-THY1 PE (1/50, Thermo Fisher), anti-CD45 PerCP-Cy5.5 (1/100, Miltenyi), anti-CD11b APC-Vio770 (1/100, Miltenyi) and anti-CD14 V500 (1/50, BD). Stained cells were analyzed using a FACS Symphony A5 instrument (BD).
3
CD39 Expression on Live HUVECs
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After flow conditioning, disassociated HUVECs were incubated with CD39-FITC (Clone A1, Biolegend) and Zombie UV viability dye (Biolegend) in Ca2+/Mg2+ free phosphate buffered saline [0.25% (v/v) foetal bovine serum] for 40 min at 4 °C. Cells were then washed twice by re-suspension. Fluorescence was measured using a LSRII flow cytometer (BD Bioscience), with the median fluorescent intensity measured on live cells.
4
Flow Cytometric Analysis of αvβ6 Integrin
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Cells were harvested by enzyme-free dissociation (CellStripper, Corning), pelleted (500 × g, 5 min), and washed once with 1× PBS. Then the cells were stained with Zombie UV viability dye (BioLegend) at RT for 15 min. The cells were pelleted and supernatants were removed, followed by incubation with PE mouse-anti-human αvβ6 (clone 10D5, BD #566922, at 1/200 × dilution in cold BD staining buffer) for 30 min on ice. The cells were re-pelleted and washed once with cold BD staining buffer. The cells were pelleted once again and resuspended in cold BD staining buffer for flow analysis on a BD Fortessa cell analyzer. Flow cytometry data were analyzed with the FlowJo software. Statistical analysis was performed with GraphPad Prism.
5
Keratinocyte Subpopulation Isolation and Sorting
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Single‐cell suspensions of freshly isolated keratinocytes were prepared from epidermal sheet as described previously and subsequently blocked with anti‐CD16/32 antibody (BioLegend). Cells were stained with APC‐conjugated CD45 and FITC‐conjugated Ly6a/Sca1 antibodies (Biolegend) for 30 min at 4°C. After incubation, cells were washed, filtered, and stained with Zombie UV™ viability dye (BioLegend) according to the manufacturer's recommendation to exclude dead cells. Keratinocytes were gated as CD45 negative live cell population and sorted to be enriched for cells of the interfollicular epithelium (IFE; Ly6a/Sca1 positive) and of hair follicles (HF; Ly6a/Sca1 negative) according to (Sakamoto et al, 2022 (link)) by a Moflo Astrios EQ cell sorter (Beckman Coulter) at cooled conditions. Sorted cells were directly pelleted and propagated for RNA isolation.
6
Multiparametric Flow Cytometry Protocol
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Single cell suspensions of LNs, PBMCs, spleens, and bone marrow were obtained. The suspensions were washed with PBS, stained with Zombie UV viability dye (BioLegend; #423108) and washed with cell staining buffer (BioLegend, #420201). The cells were stained with fluorophore-conjugated cell surface antibodies (Supplementary Table 4 –6 ). Intracellular staining was performed, relying on permeabilization and fixation reagents (BioLegend; #424401). Antibodies were arranged into appropriate panels, compensations were set up to account for fluorescent emission overlap, and the stained cells were analyzed on a Cytoflex LX flow cytometer. Storage events were gated on the population of interest, based on protocols published previously55 , and according to the gating shown in Supplementary Figures 2 , 5 , and 9 . Flow data was analyzed using FlowJo V10.
7
Multiparametric Flow Cytometry Analysis
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Cells were first incubated in 100 μL of PBS with 2 mM EDTA for 15 min with 5 µL Fc Block (Human TruStain FcX, Biolegend, cat #422301) and a 1:500 dilution of Zombie UV viability dye (Biolegend, cat# 423107). They were then washed with FACS buffer (phenol red-free DMEM, 2% FBS, 2 mM EDTA, 10 mM Hepes) and stained at 4 °C for 25 min. After cell surface staining, cells were fixed overnight at 4 °C using 100 μL of Fix/Perm buffer (eBioscience), followed by permeabilization using 1× permeabilization buffer (eBioscience) for 40 min at room temperature in the presence of intracellular antibodies. Antibody details can be found in SI Appendix, Supplementary Materials and Methods . Data were recorded on a FACSymphony flow cytometer (BD Biosciences) and analyzed using FlowJo 10 software.
8
Phenotyping Tumor-Infiltrating T Cells
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Single-cell suspensions from B16.OVA mouse tumors were stimulated with 50 ng/mL PMA (SIGMA), 1 μg/mL Ionomycin (SIGMA), Brefeldin A and Monensin (Life Technologies) for 5 hours at 37°C. Then, cells were stained with Zombie UV viability dye (Biolegend) to exclude dead cells and then surface markers were stained with the following antibodies: anti-CD8α Super Bright 702 (1/200, ThermoFisher), anti-CD45.1 Super Bright 436 (1/100, ThermoFisher), anti-CD45.2 PerCP-Cy5.5 (1/100, ThermoFisher), anti-PD1 PE (1/200, ThermoFisher), anti-Tim3 PE-Cy7 (1/200, ThermoFisher) and anti-LAG3 FITC (1/200, Thermofisher). Following surface staining, cells were fixed and permeabilized with True-Nuclear™ Transcription Factor Buffer Set (BioLegend) according to the manufacturer's instructions and intracellular staining was performed using anti-IFNγ FITC (1/100, ThermoFisher) and anti-TNF APC (1/100, ThermoFisher).
For detection of THY1 mRNA in CD14+ cells the PrimeFlow™ RNA Assay Kit (Thermo Fisher) was used following the manufacturer's instructions for 96-well plates. Validated target probes were used for THY1 (type 1 Alexa Fluor™ 647, VA1-12482-PF) and GAPDH (type 6 Alexa Fluor™ 750, VA6-10337-PF, positive control).
For detection of THY1 mRNA in CD14+ cells the PrimeFlow™ RNA Assay Kit (Thermo Fisher) was used following the manufacturer's instructions for 96-well plates. Validated target probes were used for THY1 (type 1 Alexa Fluor™ 647, VA1-12482-PF) and GAPDH (type 6 Alexa Fluor™ 750, VA6-10337-PF, positive control).
9
Cytokine Expression in Liver and Spleen Lymphocytes
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Liver and spleen lymphocyte cell suspensions were incubated in RPMI + 5% FCS containing anti-mouse CD107a mAb, Golgistop protein transport inhibitor (BD Biosciences, North Ryde, Australia) and 0.1 µg/mL peptide at 37oC in 5% CO2 for 4 h. Cells were washed in FACS buffer (2% FBS, 0.5 mM EDTA, 0.05% azide in PBS) and stained for surface markers and with ZombieUV viability dye (Biolegend, San diego, USA). Cells were washed with FACS buffer, then washed with PBS before fixing with 1% paraformaldehyde in PBS for 20 min at room temperature in the dark. Cells were washed twice with FACS buffer and incubated in FACS buffer containing 0.25% saponin (Sigma-Aldrich, St. Louis, MI, USA) and anti-mouse cytokine mAbs overnight at 4 °C in the dark. Cells were washed three times and resuspended in FACS buffer for flow cytometric analysis.
10
Isolation and Characterization of Murine NK Cells
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Mouse splenocytes obtained from naïve C57BL/6 mouse spleens were made in a single cell suspension through mechanical methods and strained through a 35 µm mesh. Then, the mouse NK Cell Isolation Kit II (MACS-Miltenyi Biotec) was used following the manufacturer’s protocol. Purified murine NK cells were collected in RPMI (10% FBS, 50 mM βME, 5 IU/ml of mIL-2 (Biolegend), and 2 ng/ml of mIL-15 (Biolegend) and used immediately for ADCC at either 1:5, 1:10, or 1:20 target to effector cell ratio (see below). Remaining NK cells were stained with 1:200 dilutions of anti-CD3, NK1.1, CD335 (NKp46), CD32/16 markers (BioLegend, catalogue number: 100221; 108709; 137611; 101323), and by 1:1000 dilution of Zombie® UV viability dye (BioLegend) and characterized by flow cytometry (BD LSRFortessa) to confirm NK cell purity and Fcγ-Receptor III expression29 (link).
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