The following anti-human fluorochrome-conjugated antibodies were used: Alexa Fluor 700-conjugated antibody to human CD3 (UCHT1); anti-IL-7Rα-BV421 (A019D5), anti-CD4-PE-Cy5 (OKT4), anti-CD45RA-APC-Cy7 (HI100), anti-Fas-BV650 (DX2), anti-CCR7-BV785 (G043H7), anti-CD31-Alexa Fluor 488 (WM59), anti-TCR γ/δ-Percp-Cy5.5 (B1), anti-CD25-BV650 (BD96; BioLegend), anti-CD19-BV711 (SJ25C1), anti-CD14-BV711 (MφP9), anti-CD3-BV510 (HIT3a), anti-CD8-Alexa Fluor 700 (RPA-T8; all from BD Biosciences); anti-CD19-PE-eFluor610 (HIB19); anti-CD14-PE-eFluor610 (61D3), and anti-CD56-APC (CMSSB; Thermo Fisher). Simultaneously, dead cells were stained using a Live/Dead Fixable Red Dead Cell Stain Kit (Thermo Fisher). The cells were fixed and permeabilized using a Foxp3 Staining Buffer Set (Thermo Fisher), and then intracellular molecules were stained (4°C, 20 minutes) using anti-Bcl-2-Alexa Fluor 488 (100; BioLegend), anti-Ki-67-PE-Cy7 (20Raj1), and anti-Foxp3-PE (236A/E7; Thermo Fisher). Detailed information about the multicolor flow cytometry panel used in this study is shown in supplemental Table 4 .
Live dead fixable red dead cell stain kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LIVE/DEAD Fixable Red Dead Cell Stain Kit is a fluorescent dye used to detect dead cells in a sample. The dye reacts with proteins in dead cells, creating a distinct red fluorescent signal that can be detected using flow cytometry or microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa flow cytometer, by BD (4 mentions)
Facscanto 2, by BD (3 mentions)
Facsdiva software, by BD (3 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (3 mentions)
Lsrii instrument, by BD (3 mentions)
7 aad, by BD (2 mentions)
Dylight 488 labeled lycopersicon esculentum lectin, by Vector Laboratories (2 mentions)
Recombinant mouse protein c, by R&D Systems (2 mentions)
Factor xa, by R&D Systems (2 mentions)
Collagenase type 4, by Worthington (2 mentions)
Pd0325901, by Fujifilm (2 mentions)
Warfarin, by Eisai (2 mentions)
Lixianar, by Eisai (2 mentions)
Prazaxa, by Eisai (2 mentions)
Recombinant mouse thrombin, by Antibodies-online (2 mentions)
Pe cy7 conjugated anti cd39, by BD (2 mentions)
Percp cy5.5 conjugated anti cd45, by BD (2 mentions)
Percp cy5.5 conjugated anti b220, by BD (2 mentions)
Pe cy7 conjugated anti cd19, by BD (2 mentions)
Apc cy7 conjugated anti cd11b, by BD (2 mentions)
39 protocols using live dead fixable red dead cell stain kit
1
Multiparametric Flow Cytometry Analysis
2
Evaluating Benzodiazepine Effects on T-cell Activation
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Example 2
To examine safety of the treatment and effect on T-cell activation, Jlat10.6 cells were treated with different benzodiazepines with and without SAHA. Forty-eight hours after incubation, cells were harvested and stained with red live/dead staining according to the manufacturer protocol (LIVE/DEAD® Fixable Red Dead Cell Stain Kit, ThermoFisher Scientific) (
In order to investigate whether alprazolam was specifically activating HIV-1 or having a general effect on T cell activation (CD4 and CD8), primary peripheral blood mononuclear cells (PBMCs) from two healthy donors were treated with 10 μM Alprazolam or 50 μM Ro5-3335, both alone or in combination with 0.5 μM SAHA. Twenty-four hours after treatment, cells were stained using Zombie yellow live and dead staining, then stained for CD3, CD8, CD4, CD69, and then fixed for analysis by flow cytometry. AntiCD3/CD28 and PHA were included as a positive control (
3
Single-cell transcriptional profiling of 22q11.21-amplified melanomas
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To obtain single-cell transcriptional portraits of 22q11.21-amplified and non-amplified tumors, we analyzed a primary acral melanoma specimen (YUJASMIN) with six focal copies of 22q11.21, as determined by WES, and three primary acral melanomas without amplification of 22q11.21 (YUGRUS, YUMASK, YUPARK; Supplementary Data 1 ). For YUJASMIN, the 10x Chromium 5′ expression profiling platform with V1 chemistry was applied to a cryopreserved tumor cell suspension sorted for viable singlets to target 10,000 cells (Supplementary Fig. 10 ). Cells were sorted in the following ratios prior to library preparation: 50% CD3+CD45+ T cells: 25% CD3–CD45+ non-T immune cells: 25% CD45– stromal/cancer cells. Cell viability was assessed by the LIVE/DEAD™ Fixable Red Dead Cell Stain Kit (catalog no. L34971, Thermo Fisher). The following antibodies were used: Alexa Fluor® 488 anti-human CD45 antibody (clone H130, catalog no. 304019, BioLegend); APC anti-human CD3 antibody (clone HIT3a, catalog no. 300319, BioLegend) (Supplementary Data 9 ). The 10x library was sequenced on an Illumina HiSeq 2500 instrument. For the other three tumors, the 10x Chromium 5′ assay with V2 chemistry was applied to cryopreserved tumor cell suspensions and sequenced on an Illumina NovaSeq 6000 instrument.
4
Multiparametric Flow Cytometry of Blood, Spleen, and Tumor
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Whole blood, spleen, and tumor tissues were processed for flow cytometry analysis. In brief, gentleMACS Octo Dissociator from Miltenyi Biotec was used for spleen and tumor-tissue dissociation, followed by filtering through 100 µm screens. After tissue dissociation, the cells were washed with PBS and incubated with the antibody panel for 15–20 min at room temperature. The red blood cells were lysed by Pharm Lyse (BD Biosciences, San Jose, CA, USA) for 8–12 min following antibody staining at room temperature. Live or dead cells were detected by LIVE/DEAD Fixable Red Dead Cell Stain Kit (Thermo Fisher Scientific). Samples were centrifuged and washed with PBS before suspension in PBS for data acquisition on BD FACSCanto II using FACSDiva software (BD Biosciences).
Fluorochrome–conjugated mAb to the following human antigens were used: CD45-V510 (clone HI30), CD45-phycoerythrin (PE; clone HI30), CD3-FITC (clone UCHT1), CD4-PE-cyanine 7 (clone SK3), CD8-allophycocyanin-cyanine 7 (clone SK1), CD19-allophycocyanin (clone HIB19), PD-1-PE (clone EH12.2H7), and PD-L1-V421 (clone 29E.2A3). All antibodies were purchased from BioLegend.
Fluorochrome–conjugated mAb to the following human antigens were used: CD45-V510 (clone HI30), CD45-phycoerythrin (PE; clone HI30), CD3-FITC (clone UCHT1), CD4-PE-cyanine 7 (clone SK3), CD8-allophycocyanin-cyanine 7 (clone SK1), CD19-allophycocyanin (clone HIB19), PD-1-PE (clone EH12.2H7), and PD-L1-V421 (clone 29E.2A3). All antibodies were purchased from BioLegend.
5
Visualizing Dead Cells with Fixable Dye
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The dead cells were visualized using the LIVE/DEAD Fixable Red Dead Cell Stain kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Images were obtained using a confocal microscope (Leica TCS SP5 Microsystems, Wetzlar, Germany).
6
Quantifying SN-MM Cell Viability
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SN-MM cells were detached using the Tryple Express Enzyme (cat.n. 12604013, Thermo Fisher Scientific), washed in 1 mL cold DPBS w/o proteins and stained using Live/Dead Fixable Red Dead Cell Stain Kit (cat. n. L23102, Life Technologies) and anti-CD271 (clone REA844) PE-conjugated antibody (cat.n. 130-112-601, Miltenyi Biotec, Bergisch Gladbach, Germany) following manufacturer’s instructions. Unstained cells were used as negative controls. Results were reported as the percentage of positive cells after gating on single live cells. Samples were acquired with MACS Quant® Analyzer 16 (Miltenyi Biotec) and results were analyzed by FlowJo X software v10.8 (Tree Star Inc, Wilmington, NC, USA).
7
Cell Viability Assay via LIVE/DEAD Staining
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Cell viability was determined using the LIVE/DEAD Fixable Red Dead Cell Stain Kit (Thermo Fisher Scientific, L34972). Briefly, cells were harvested, washed, stained, and fixed according to the manufacturer’s guidelines. R-phycoerythrin signal was detected by a FACSort apparatus (Becton Dickinson) and analyzed using FlowJo software.
8
Quantifying CD206 and CD80 Expression in THP-1 Cells
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After staining the THP-1 cells in an Invitrogen LIVE/DEAD Fixable Red Dead Cell Stain kit (#L23102, Thermo Fisher Scientific Inc., Waltham, MA, USA), viable cells were blocked using 10μg/mL human immunoglobulin G (IgG) diluted in 0.5% bovine serum albumin (BSA)/1mM, ethylenediaminetetraacetic acid (EDTA)/0.01%, sodium azide staining buffer on ice for 10 min, and then incubated with anti-human CD206 (MMR)-PE antibody (#12-2069-41, 1:50, eBioscience, Thermo Fisher Scientific Inc., Waltham, MA, USA) on ice for 1 h, before cell fixation in 2% paraformaldehyde (PFA) on ice for 15 min. Next, the cells were incubated with anti-human CD80 (B7-1) Fluorescein isothiocyanate (FITC) antibodies (#11-0809-41, 1:50, eBioscience, Thermo Fisher Scientific Inc., Waltham, MA, USA) in 0.5% saponin on ice for 45 min, washed with staining buffer, and then flow-cytometry data acquisition was performed using the BD FACSCanto II system (BD Biosciences, Thermo Fisher Scientific Inc. Waltham, MA, USA). Data obtained were analyzed using Flowjo software v. 9.6.2 (FlowJo, LLC, Tree Star, Inc., Ashland, OR, USA).
9
Visualizing Tissue Hypoxia Profiles
10
Flow Cytometric Phenotyping of Immune Cells
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Cells were stained with antibodies against cell-surface molecules for 20 min at 4°C. The LIVE/DEAD Fixable Red Dead Cell Stain Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used to identify live cells. For intracellular staining, cells were fixed and permeabilized with the Foxp3 fixation/permeabilization solution (ThermoFisher Scientific) for 20 min at 4°C, and stained with antibodies against intracellular molecules for 20 min at 4°C. The stained cells were analyzed using FACS CytoFLEX (Beckman Coulter, Brea, California, USA) and the data were analyzed using FlowJo software V.10 (Treestar, San Carlos, California, USA). The antibodies used are listed in online supplemental table 1 and the gating strategies for flow cytometry are shown in online supplemental figure 1 . Expression of PD-1 on CD8+ T lymphocytes from peripheral blood was used to gate for PD-1neg, PD-1int, and PD-1high populations.
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