Cells were stained with one of three staining combinations in four different experiments as detailed below. Experiments 1, 2, and 3 were performed with K562 cells (ATCC® CCL243™). In Experiment 1, all cells were stained with DNA stains - Hoechst 33342 “ThermoFisher” (2 µM final concentration, 20 minutes at room temperature) and propidium iodide “ThermoFisher” (3.75 µM final concentration, 20 minutes at room temperature) - where Hoechst permeates all cells and propidium iodide enters dead cells only. In Experiments 2, and 3 - half the cells were stained with CellTracker Green CMFDA “ThermoFisher” (1 µM final concentration, 30 minutes at room temperature) and LIVE/DEAD® Fixable Red Dead Cell Stain “ThermoFisher” (per manufacturer protocol), while the other half were stained with CellTracker Orange CMRA “ThermoFisher” (1 µM final concentration, 30 minutes at room temperature) and LIVE/DEAD® Fixable Red Dead Cell Stain “ThermoFisher” (per manufacturer protocol). CellTracker Orange and CellTracker Green stain all cells, whereas Fixable Red Dead Cell enters only dead cells. Post-staining, cells were thoroughly washed with PBS to remove unincorporated stain and re-suspended in PBS or 1x DSP for fresh and fixed cells respectively.
Live dead fixable red dead cell stain
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Live/Dead Fixable Red Dead Cell Stain is a fluorescent dye used to identify dead cells in a sample. It labels dead cells with a red fluorescent signal, allowing for their detection and separation from live cells.
Automatically generated - may contain errors
Lab products found in correlation
Diva software, by BD (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
Anti cd3 af700, by BioLegend (2 mentions)
Anti cd4 pb, by BioLegend (2 mentions)
Lsr 2 flow cytometer, by BD (2 mentions)
7 aminoactinomycin d 7 aad, by Merck Group (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
7 aminoactinomycin d, by Merck Group (1 mentions)
Affinipure f ab 2 fragment donkey anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Anti cd3ε fitc 145 2c11, by BD (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
7 aad, by BD (1 mentions)
Facscan, by BD (1 mentions)
Macsquant, by Miltenyi Biotec (1 mentions)
Human fc block, by BD (1 mentions)
Lsr 2 cytometer, by BD (1 mentions)
Ultracomp ebeads, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Alexa fluor555 azide, by Thermo Fisher Scientific (1 mentions)
16 protocols using live dead fixable red dead cell stain
1
Multicolor Cell Staining Optimization
2
Flow Cytometric Analysis of Btnl-Transfected Cells
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Btnl-transfected cells were stained with APC-conjugated rabbit anti-FLAG or anti-HA antibody (PerkinElmer). For detection of intracellular expression of FLAG-Btnl6, cells were permeabilized using the cytofix/cytoperm kit (BD Biosciences). CFSE-labeled cells were stained with Alexa Fluor 700-conjugated anti-CD45 (eBioscience), eFluor450-conjugated anti-pan TCRγδ (GL3, eBioscience), APC or APC-Cy7-conjugated anti-TCRb (eBioscience), PerCPCy5.5-conjugated anti-CD25 (eBioscience), PE-conjugated anti-TCR Vγ1.1/Cr4 (BioLegend), eFluor660-conjugated anti-TCR Vδ4 (eBioscience), anti-TCR Vγ7 (kindly provided by Dr. Pablo Pereira, Institut Pasteur, Paris, France), and 7-aminoactinomycin D (7AAD; Sigma-Aldrich) or LIVE/DEAD® Fixable Red Dead Cell Stain (Molecular Probes®, Life Technologies). Cells were gated on 7AAD or LIVE/DEAD® Fixable Red negative cells to exclude non-viable cells. Cells were acquired on LSR II flow cytometer using the DIVA software (BD Biosciences), and analysis of data was performed using the FlowJo Software version 7.6.5.
3
Characterization of Btnl1 Cell Surface Expression
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Cell-surface antigen expression was analyzed using the following antibodies: anti-Btnl1 rabbit polyclonal antiserum, pre-immune serum, anti-CD45-Alexa Fluor 700 (30-F11; eBioscience, San Diego, CA), anti-CD3ε-FITC (145–2C11; BD PharmigenTM, San Diego, CA), anti-pan TCRγδ-eFluor450 (eBioGL3; eBioscience, San Diego, CA), anti-TCRβ-APC (H57–597; eBioscience, San Diego, CA), anti-TCR Vγ1.1/Cr4-PE (2.11; BioLegend, San Diego, CA), anti-TCR Vδ4-eFluor660 (GL2; eBioscience, San Diego, CA), anti-TCR Vγ7-biotin (kindly provided by Dr Pablo Pereira, Institut Pasteur, Paris, France), 7-aminoactinomycin D (7AAD; Sigma-Aldrich, St. Louis, MO), and LIVE/DEAD® Fixable Red Dead Cell Stain (Molecular Probes®, Life Technologies, Eugene, OR). Detection of anti-Btnl1 and anti-TCR Vγ7 was achieved with APC-conjugated AffiniPure F(ab’)2 fragment donkey anti-rabbit IgG (H+L) (Jackson ImmunoResearch, West Grove, PA) and streptavidin-APC-CyTM7 (BD Biosciences, San Diego, CA), respectively. Cells were gated on 7AAD or LIVE/DEAD® Fixable Red negative cells to exclude non-viable cells, and positive staining for Btnl1 was determined by comparison with pre-immune serum. Cell samples were acquired on LSR II flow cytometer using the DIVA software (BD Biosciences, San Diego, CA), and analysis of data was perfomed using the FlowJo Software version 7.6.5 (Ashland, OR).
4
Mitochondrial ROS and Cell Death Analysis
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Cells were detached using ice cold PBS containing 2 mM EDTA and stained for mitochondrial ROS (MitoSOX Red, Life Technologies) or cell death (7-AAD, eBioscience or LIVE/DEAD fixable red dead cell stain, Life Technologies). Proportions of mitochondrial ROS producing (MitoSOX+) or dead (7AAD+ or LIVE/DEAD+) cells were determined in infected (GFP+) and uninfected (GFP-) cell populations by flow cytometry (FACScan, BD or MACSQuant, Miltenyi Biotec). Data analysis was done using FlowJo software (Tree Star).
5
PBMC and Monocyte Staining Protocol
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Following recovery from culture, PBMCs or monocytes were washed once in ice cold PBS prior to staining with Live/Dead fixable red dead cell stain (Life Technologies, Carlsbad, CA) for 30 m at 4 °C. Cells were washed once in cold PBE (PBS with 0.5% BSA and 2 mM EDTA) and stained with antibodies for extracellular markers diluted in PBE for 30 m at 4 °C. For ICS, cells were treated with Cytofix/Cytoperm (BD Biosciences, San Jose, CA) for 20 m at 4 °C, washed, and stained with antibodies for intracellular markers at 1:100 for 30 m at 4 °C. Human Fc Block (BD Biosciences) was used in all staining buffers. For intracellular cytokine staining, Brefeldin A (Millipore Sigma) was added to the culture at 5 mg/mL 6 h prior to harvest. Single-stained controls were prepared using UltraComp eBeads (Invitrogen, Carlsbad, CA). Data was acquired on a BD LSR II cytometer (BD Biosciences). Compensation and gating were accomplished using FlowJo v10 (TreeStar, Ashland, OR). Gating strategies are included in the supplement (Supplementary Fig. 7 ). A complete list of conjugated clones may be found in Supplementary Table 2 .
6
Murine Bone Marrow and Thymus Cell Staining
7
Multiparametric Flow Cytometry of iPSCs and iPSC-CMs
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iPSCs were pulsed with 20 µM EdU for 1 h using the Click-iT EdU Alexa Fluor 647 Flow kit (Thermo Fisher C10424). iPSC-CMs were pulsed with 10 µM EdU for 24 h. Cells were stained with LIVE/DEAD Fixable Red Dead Cell Stain (Thermo Fisher L23102) using the manufacturer’s protocol before fixing in 4% paraformaldehyde. Click-iT labeling of EdU was performed using the manufacturer’s protocol. iPSCs were stained with OCT4 (Millipore MAB4419; 1:100 dilution) and iPSC-CMs were stained with cTnT (Thermo Fisher MA5-12960, 1:200 dilution) for 1 h at room temperature. Cells were incubated with goat-anti-rabbit Alexa 488 (Jackson Immuno Research 111-546-045, 1:250 dilution) for 30 min at room temperature. DNA was stained with FxCycle Violet Flow Reagent (Thermo Fisher R37166) using the manufacturer’s protocol. Four-color flow cytometry analysis was performed on the SONY SH800 using unstained samples and fluorescence minus one controls to set detectors. FlowJo v10 was used to analyze flow cytometry data.
8
RHAMM Expression in Viable Dendritic Cells
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Four hours after electroporation (healthy volunteer donor samples) or two hours after thawing (patient samples), DCs were stained with LIVE/DEAD® Fixable Red Dead Cell Stain (cat# L23102, Thermo Fisher Scientific) prior to fixation and permeabilization for intracellular staining using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit (cat# 554714, BD Biosciences) according to the manufacturer's instructions. Subsequently, samples were incubated with mouse anti-RHAMM monoclonal antibody (mAb; clone 2D6, cat# MO20030, Neuromics) or isotype control mouse IgG1 mAb (cat# 349040, BD Biosciences) followed by PE-labeled rat anti-mouse IgG1 mAb (clone X56, cat# 340270, BD Biosciences). RHAMM expression in viable (LIVE/DEAD−) cells was determined on a FACScan flow cytometer (BD Biosciences).
9
Cell Membrane Integrity Analysis
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For analysis of cell membrane integrity, iCell® Cardiomyocytes were grown on cover slips, infected with MOI 10 for 8 h. Cells were fixed in 4% paraformaldehyde in PBS for 15 min, and subsequently stained with LIVE/DEAD Fixable Red Dead Cell Stain (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.
10
Quantifying Lectin-Mediated Nucleic Acid Uptake
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DCs or Raji cells stably expressing DC-SIGN or Langerin [41 (link)] were incubated in the presence or absence of EGTA (10 mM) or α-DC-SIGN (20 μg/ml, clone AZN-D1) for 30 min followed by ODN-2216-FITC (Invivogen) or EC-DNA-FITC (FastTag Basic Nucleic Acid Labeling Kit, Vector Laboratories) for 10 min at 37°C. Dead cells were excluded using LIVE/DEAD™ Fixable Red Dead Cell Stain (Thermo Fisher). Cells were analyzed on a FACS Calibur (BD).
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