To detect the distribution of immune cells, we stained 1 × 106 single cells with a Live/Dead Stain (1:1000; BD Biosciences, San Jose, CA, USA), Pacific blue-conjugated CD3 (1:200; BD Biosciences, San Jose, CA, USA), FITC-conjugated CD4 (1:200; BD Biosciences, San Jose, CA, USA), APC-conjugated CD8 (1:200; BD Biosciences, San Jose, CA, USA), APC-Cy™7-conjugated CD45 (1:200; Thermo Scientific, Waltham, MA, USA), FITC-conjugated CD11b (1:200; Thermo Scientific, Waltham, MA, USA), Pacific blue-conjugated MHC II (1:200; BioLegend, San Diego, CA, USA), and PE-conjugated CD86 (1:200; BD Biosciences, San Jose, CA, USA) antibodies for 30 min at 4 °C without light. The samples were processed on a BD FACSCanto II (BD Biosciences, San Jose, CA, USA), and all data were analyzed with FlowJo v10 software (Tree Star).
Live dead stain
Manufactured by BD
Sourced in United States
The Live/Dead stain is a fluorescent-based reagent that is used to assess the viability of cells in a sample. It can distinguish between live and dead cells by selectively staining them with different fluorescent dyes, providing a rapid and quantitative method for evaluating cell health and integrity.
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Lab products found in correlation
Rat anti cd16 cd32 fc antibodies, by BD (2 mentions)
Rat anti cd11b pecy7 antibodies, by Thermo Fisher Scientific (2 mentions)
Rat anti cd45 pecpcy5.5 antibodies, by Thermo Fisher Scientific (2 mentions)
Mouse glia panel, by NanoString (2 mentions)
Facsaria 2, by BD (2 mentions)
Live dead stain, by Thermo Fisher Scientific (2 mentions)
Pe conjugated cd86, by BD (1 mentions)
Apc conjugated cd8, by BD (1 mentions)
Fitc conjugated cd4, by BD (1 mentions)
Fitc conjugated cd11b, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Monensin, by Merck Group (1 mentions)
Cd3 pe cy7, by Thermo Fisher Scientific (1 mentions)
Cd56 ecd, by Beckman Coulter (1 mentions)
Tnf α fitc, by BD (1 mentions)
Cd16 apc cy7, by BD (1 mentions)
Ifnγ v450, by BD (1 mentions)
Rhil 12, by Miltenyi Biotec (1 mentions)
Rhil 18, by R&D Systems (1 mentions)
Ionomycin, by Merck Group (1 mentions)
7 protocols using live dead stain
1
Immune Cell Profiling by Flow Cytometry
2
Microglia Isolation and Transcriptomic Profiling
3
Cell Viability Assessment in 3D and 2D
4
Cytokine Production in PBMCs
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For intracellular staining for IFN-γ production, PBMCs were incubated with 50 ng/ml of rhIL-12 (Miltenyi Biotec) and 50 ng/ml of rh-IL18 (R&D Systems) for 21 h at 37°C. One mM monensin (Sigma-Aldrich, Gillingham, UK) was added for the final 3 h. Cells were stained with antibodies to CD3-PE-Cy7 (eBioscience), CD16-APC-Cy7 (BD Biosciences) and CD56-ECD (Beckman Coulter) and subsequently fixed and permeabilized, followed by intracellular staining for IFN-γ-V450 (BD Biosciences). Dead cells were excluded by live/dead stain (Invitrogen).
For TNF-α production, PBMCs were stimulated with phorbol myristate acetate (PMA) (3 ng/ml) and ionomycin (100 ng/ml) (Sigma-Aldrich) for 3 h in the presence of 1 mM of monensin (Sigma-Aldrich). Cells were stained with antibodies to CD3-PE-Cy7 (eBioscience), CD16-APC-Cy7 (BD Biosciences) and CD56-ECD (Beckman Coulter) in the presence of live/dead stain followed by fixing, permeabilization and intracellular staining for TNF-α-FITC (BD Biosciences).
For TNF-α production, PBMCs were stimulated with phorbol myristate acetate (PMA) (3 ng/ml) and ionomycin (100 ng/ml) (Sigma-Aldrich) for 3 h in the presence of 1 mM of monensin (Sigma-Aldrich). Cells were stained with antibodies to CD3-PE-Cy7 (eBioscience), CD16-APC-Cy7 (BD Biosciences) and CD56-ECD (Beckman Coulter) in the presence of live/dead stain followed by fixing, permeabilization and intracellular staining for TNF-α-FITC (BD Biosciences).
5
Microglia Isolation and Transcriptomic Profiling
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Microglia were isolated using the Percoll gradient and washed with cold FACS buffer as described for the ex vivo phagocytic assay except that cell pellet was resuspended in 300 μl of cold FACS buffer and blocked with rat- anti CD16/CD32 Fc antibodies 1:100 (Cat# 553142, BD Biosciences) and live/dead stain (Cat. #34965, Thermo Fisher) for 10 min on ice. Microglia were then stained with rat-anti CD11b-PeCy7 antibodies 1:100 (Cat# 25-0112-82, ebiosciences) and rat-anti CD45-PeCPCy5.5 antibodies 1:100 (Cat# 45-0451-82, ebiosciences) for 25 min on ice. Labeled cells were washed with 3 ml of cold FACS buffer, spun as above, resuspended in 300ul of cold FACS buffer and sorted using BD FACSAria™ II flow cytometer (BdBiosciences). Sixty thousand CD11b+, CD45 low cells were collected per sample and spun down at 400 g for 8 min at 4 °C. The supernatant was discarded, and the pellet resuspended in 5ul of 1/3RLT buffer (cat. No. 79216, Qiagen) and frozen at —80 °C until further genomic analysis using the mouse Glia panel (Nanostring, Cat. # XT-Mm Glial profiling CSO). Five μl of lysate were used for hybridization setup (65 °C for 16hrs) and mRNA counting using the nCounter manufacturer protocol. Samples from 8 pups (from 4 to 5 independent litters) per rearing and sex group were run in 4 batches of 12 reactions each (total of 48 reactions).
6
Treg, Th1, and Th17 Cell Analysis by Flow Cytometry
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To determine Treg frequencies, single cell suspensions prepared as above were blocked with rat anti-mouse CD16/CD32 Fc block (BD Pharmingen, San Jose, CA, USA), stained with cell surface antibodies, then fixed and permeabilized with the FoxP3 staining buffer kit (eBioscience, San Diego, CA, USA) before staining with any intracellularly directed antibodies. The following anti-mouse antibody panel was used: CD25-PE, Helios-efluor450, Foxp3-APC, CD4-FITC, and Live/Dead stain conjugated to efluor780 (all eBioScience). For Th1 and Th17 cell detection, single cell suspensions were stimulated overnight (16 hours) with 500 ng/mL phorbal myristate acetate (PMA) and 2 μg/mL ionomycin (I) in culture media (10% FBS, RPMI, penicillin, streptomycin, β-mercaptoethanol) at 37°C in the presence of GolgiStop (BD Biosciences, San Jose, CA, USA) for the last 4 hours. Cells were then fixed and permeabilized using an intracellular cytokine detection kit (BD Biosciences). They were stained with the following anti-mouse antibody panel: CD4-PE-Cy7, interferon-γ (IFN-γ)-FITC (BD Biosciences), TNF-α-PE (eBioscience), IL-17A-AlexaFluor647 (BD Pharmingen), and Live/Dead stain as above. Flow cytometric data were acquired on a LSRFortessa cell analyzer (BD Biosciences).
7
Multiparameter Flow Cytometry Profiling of PBMCs
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PBMCs from HIV-infected or HIV-negative patients were incubated with fluorochrome-conjugated antibodies for at least 15–20 minutes at 4°C or on ice, protected from light. The following fluorochrome-conjugated anti-human antibodies were used: CD3 (HIT3α), CD4 (RPA-T4), CD10 (HI10a), CD20 (2H7), CD25 (BC96), CD27 (O323), CD38 (HIT2), PD-1 (EH12.2H7), and CXCR3 (G025H7) were all from BioLegend. CD19 (HIB19), CXCR5 (RF8 B2), ICOS (DX29) and CD21 (B-ly4) were from BD Biosciences and CD45RA (2H4LDH11LDB9) from Beckman Coulter. LIVE/DEAD Fixable Dead Cell Stain (Life Technologies) was used to gate on live cells; and in some cases both LIVE/DEAD stain and Annexin V (BD Biosciences) were used. Samples were acquired on a BD LSR II.
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