Blood was collected in sodium heparin tubes and processed within 3 h of collection. The whole blood assay was adapted from the protocol described by Hanekom et al.46 (link) Briefly, 0.5 ml of whole blood was stimulated with a pool of 300 Mtb-derived peptides (Mtb300, 2 µg/ml)47 (link) at 37 °C for 5 h in the presence of the costimulatory antibodies, anti-CD28 and anti-CD49d (1 μg/ml each; BD Biosciences) and Brefeldin-A (10 µg/ml; Sigma-Aldrich). Unstimulated cells were incubated with costimulatory antibodies and Brefeldin-A only. Red blood cells were then lysed in a 150 mM NH4Cl, 10 mM KHCO3, and 1 mM Na4EDTA solution. Cells were stained with a Live/Dead Near-InfraRed dye (Invitrogen), and then fixed using a Transcription Factor Fixation buffer (eBioscience), cryopreserved in freezing media (50% fetal bovine serum, 40% RPMI, and 10% dimethyl sulfoxide) and stored in liquid nitrogen until use.
Live dead near infrared dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Live/Dead Near-InfraRed dye is a fluorescent dye used to distinguish between live and dead cells in a sample. It functions by differentially staining live and dead cells, allowing for their identification and quantification under near-infrared fluorescence imaging.
Automatically generated - may contain errors
Lab products found in correlation
Transcription factor fixation buffer, by Thermo Fisher Scientific (3 mentions)
Brefeldin a, by Merck Group (3 mentions)
Anti cd28 and anti cd49d, by BD (2 mentions)
Cd27 pe cf594 m t271, by BD (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Anti cd49d, by BD (1 mentions)
Anti cd28, by BD (1 mentions)
Nuclear factor fixation and permeabilization buffer set, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (1 mentions)
Cd3 apc cy7 sk7, by BioLegend (1 mentions)
Cd16 apc cy7 3g8, by BioLegend (1 mentions)
Streptavidin bv421, by BioLegend (1 mentions)
Cd21 pe b ly4, by BD (1 mentions)
Facsaria 3, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Annexin 5 apc, by BD (1 mentions)
7 protocols using live dead near infrared dye
1
Tuberculosis Antigen-Specific T-Cell Assay
2
Whole Blood Assay for Mtb-Specific T-cells
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Blood was collected in sodium heparin tubes and processed within 3 h of collection. The whole blood assay was adapted from the protocol described by Hanekom et al.51 Briefly, 0.5 mL of whole blood was stimulated with a pool of 300 Mtb‐derived peptides (Mtb300, 2 µg mL−1)52 at 37°C for 5 h in the presence of the co‐stimulatory antibodies, anti‐CD28 and anti‐CD49d (1 μg mL−1 each; BD Biosciences, San Jose, CA, USA) and Brefeldin‐A (10 µg mL−1; Sigma‐Aldrich, St Louis, MO, USA). Unstimulated cells were incubated with co‐stimulatory antibodies and Brefeldin‐A only. Red blood cells were then lysed in a 150 mm NH4Cl, 10 mm KHCO3 and 1 mm Na4EDTA solution. Cells were stained with a Live/Dead Near‐InfraRed dye (Invitrogen, Carlsbad, CA, USA) and then fixed using a Transcription Factor Fixation buffer (eBioscience, San Diego, CA, USA), cryopreserved in freezing media (50% foetal bovine serum, 40% RPMI and 10% dimethyl sulfoxide) and stored in liquid nitrogen until use.
3
Whole Blood Assay for Tuberculosis
4
Murine Thymocyte Apoptosis Kinetics
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Murine thymocytes were collected by smashing the thymus in a 40-μm cell strainer. Cells were suspended in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 2 mM l -glutamine at 1 × 106 cells/ml and cultured for 0, 3, 6, 12, 18, or 24 hours. Thymocytes were then incubated with anti-Thy1.2 antibody and a fixable LIVE/DEAD near-infrared dye (Thermo Fisher Scientific). After two washes, the cells were stained with 5 μl of phycoerythrin–annexin V in 100 μl of binding buffer containing 0.01 M Hepes (pH 7.4), 0.14 M NaCl, and 2.5 mM CaCl2 for 15 min. An additional 400 μl of binding buffer was added to the suspension before analysis.
5
Intracellular Flow Cytometry Staining
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Subsequent to cytokine treatment, cells were washed and cultured for an additional 4 h in cytokine-free cDMEM containing monensin. Intracellular flow cytometry staining was performed according to standard procedures for the Nuclear Factor Fixation and Permeabilization Buffer set (BioLegend). Live/Dead Near-Infrared dye (Thermo Fisher) was used to determine viability. Transduction efficiency was detected by EGFP expression and tetramer staining (Supplementary Fig. 2 ). CTLs were analyzed using a LSRFortessa (BD Biosciences) and FlowJo Software (TreeStar).
6
Phosphorylation Analysis of PBMCs
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PBMCs were rested after thawing for 4 h in RPMI 1640 medium (Thermo Fisher Scientific) without supplements and subsequently incubated in RPMI 1640 containing live/dead near-infrared dye (Life Technologies) for 20 min at room temperature. Cells were washed, split into two wells, and resuspended in either only RPMI 1640 for the unstimulated sample or RPMI 1640 containing 10 µg/ml goat anti-human IgG/IgA/IgM f(ab)2 (Jackson ImmunoResearch). Stimulation was done for 5 min including a 2-min centrifugation step to remove the medium, followed by addition of the fixation and permeabilization solution of the Foxp3 staining kit according to the manufacturer’s instructions (eBiosciences). Cells were fixed and permeabilized for 45 min at 4°C and then stained with antibodies including CD3 APC-Cy7 (SK7; BioLegend), CD10 PE-Cy5 (HI10a; BD Biosciences), CD14 APC-Cy7 (HCD14; BioLegend), CD16 APC-Cy7 (3G8; BioLegend), CD19 BV510 (SJ25C1; BD Biosciences), CD21 BV711 (B-ly4; BD Biosciences), CD27 PE-CF594 (M-T271; BD Biosciences), IgD APC (IA6-2; BioLegend), and total phosphor-tyrosine PE (PY20; BD Biosciences) for 30 min at room temperature. Cells were extensively washed and measured using a BD Fortessa. Analysis was done with FlowJo 10 (TreeStar).
7
Apoptosis Profiling of B Cell Subsets
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To determine the frequency of apoptotic cells, we analyzed two major events during apoptosis using flow cytometry: cleavage of caspase 3 and the exposure of phosphatidylserine on the outer leaflet of the cell membrane (Segawa et al., 2014 (link)). Cryopreserved PBMCs from 10 healthy controls and 10 chronically infected patients were analyzed for the frequency of apoptotic cells within the different B cell subsets (Table S2). PBMCs were stained for 30 min with antibodies against surface markers including CD19 biotin (HIB19; BD Biosciences), CD21 PE (B-ly4; BD Biosciences), CD27 PE-CF594 (M-T271; BD Biosciences), IgD PE-Cy7 (IA6-2; BioLegend), and the apoptosis markers cleaved caspase 3 inhibitor FITC-C6-DEVD-FMK (AAT Bioquest). Subsequently, live/dead near-infrared dye (Life Technologies) and streptavidin BV421 (BioLegend) staining was performed for 30 min followed by staining with Annexin V APC (BD Biosciences) according to the manufacturer’s protocol. Samples were analyzed on either a FACS Aria III or a BD FACS Verse immediately after staining and analyzed with FlowJo version 10. Apoptotic cells were defined as double positive for Annexin V and FITC-C6-DEVD-FMK (Jayaraman, 2003 (link)) after dead cell exclusion using live/dead near-infrared dye.
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