Isolated microglia samples were first treated with Fc blocker (BD Pharmingen), then stained with surface CX3CR1-APC (1:500, Biolegend) and LIVE/DEAD Fixable Near-IR Dead Cell Staining Kit (1:2000, Invitrogen). The cells were then washed, fixed, and permeabilized with FoxP3/Transcription factor staining set (ThermoFisher), and then stained with Ki67–PerCP–eFluor710 (1:2000, Invitrogen). Flow cytometry was performed with a BD FACS Canto II and the results were analyzed by Flowjo software v10.6.1. Our gating strategy is similar as previously reported55 and presented in Supplementary Fig. S3 .
Live dead fixable near ir dead cell staining kit
Manufactured by Thermo Fisher Scientific
The LIVE/DEAD fixable Near-IR Dead Cell staining kit is a fluorescent dye-based reagent used to label and detect dead cells in a sample. It binds to proteins in compromised cell membranes, providing a quantitative measure of cell viability.
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Lab products found in correlation
Mhcii 1 a 1 e, by BioLegend (2 mentions)
Mpdca1, by BioLegend (2 mentions)
Siglec h, by BioLegend (2 mentions)
Anti mouse cd11c, by BioLegend (2 mentions)
Pe conjugated anti mouse ly49q, by MBL Life Science (2 mentions)
Cd11b, by BioLegend (2 mentions)
Fc blocker, by BD (1 mentions)
Foxp3 transcription factor staining set, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Ultracomp ebeads, by Thermo Fisher Scientific (1 mentions)
Rat anti mouse cd16 cd32 fc block, by BD (1 mentions)
Anti cd16 bv785, by BioLegend (1 mentions)
Fluorofix buffer, by BioLegend (1 mentions)
Anti cd3 percp cy5.5 clone ucht1, by BioLegend (1 mentions)
Anti cd19 apc cy7, by BioLegend (1 mentions)
Anti cd56 buv395, by BD (1 mentions)
Cellfix, by BD (1 mentions)
Perm wash, by BD (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
8 protocols using live dead fixable near ir dead cell staining kit
1
Quantifying Microglia Proliferation by Flow
2
Neutrophil Identification Workflow
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BAL cells were stained with the Live/Dead Fixable Near-IR-Dead Cell staining kit (Invitrogen) for 20 minutes in PBS prior to blockade with anti-CD16/CD32 Fc receptor block (BD Pharminogen) for 20 minutes. Cells were then washed in PBS containing 0.1% sodium azide and 1% BSA followed by staining for surface markers at 40C for 30 minutes. Details of antibodies used are shown in Supplementary Table 1 . Cells were subsequently washed in PBS containing 0.1% sodium azide and 1% BSA before fixation in 2% paraformaldehyde. Neutrophils were identified using the following surface markers, as previously described60 (link): The gating strategy adopted is shown in Supplementary Fig. 5 .
3
Quantitative Immune Cell Profiling
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Single-cell suspensions of total splenocytes pre-chemotaxis and following transmigration through the HTS Transwell®-96 pores in the absence or presence of stimuli were pelleted and washed in cold PBS. Cells were stained with the LIVE/DEAD Fixable Near IR Dead cell staining kit (Invitrogen) for 10 min at RT while shielded, and washed with flow cytometry buffer (PBS + 0.1% sodium azide + 1% BSA) and pelleted. Cells were then stained with rat anti-mouse CD45-BV711, rat anti-mouse CD3-PE-Cy7 (eBioscience), rat anti-mouse CD19-FITC, rat anti-mouse CD11b-Alexa Fluor 700, rat anti-mouse F4/80-PE, rat anti-mouse Gr-1-APC, and purified rat anti-mouse CD16/CD32 Fc Block™ (BD Biosciences) for 30 min at 4°C in flow cytometry buffer and then fixed in 1% paraformaldehyde. Sample acquisition was performed over 2 min per sample using a 5-laser BD LSR Fortessa III instrument (BD Biosciences), and all samples were kept as individuals and not pooled. Instrument standardization and calibration were performed according to the manufacturer’s instructions. Compensation was accounted for using UltraComp eBeads™ (Thermo Fisher Scientific). Immune cell phenotyping was analyzed with FlowJo software (Version 10.9; BD Biosciences).
4
Comprehensive NK Cell Phenotyping
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PBMCs from healthy and untreated HIV-1-infected individuals were gently thawed by adding dropwise complete medium supplemented with 25 U/ml Benzonase Nuclease (Merck Milipore Novagen) followed by a 30 min incubation at 37°C and 5% CO2. After counting and washing with PBS, cells were incubated with LIVE/DEAD fixable Near-IR Dead Cell staining kit (Invitrogen) and the following antibodies at 4°C: anti-CD3-PerCP-Cy5.5 (clone UCHT1, BioLegend), anti-CD4-BV650 (clone RPA-T4, BioLegend), anti-CD8-AF700 (clone EB6B, BioLegend), anti-CD14-APC-Cy7 (clone HCD14, BioLegend), anti-CD19-APC-Cy7 (clone HIB19, BioLegend), anti-CD56-BUV395 (clone NCAM16.2, BD Optibuild), anti-CD16-BV785 (clone 3G8, BioLegend), anti-CD57-BV510 (clone QA17A04, BioLegend), anti-NKG2A-PE-Vio615 (clone REA110, Miltenyi), anti-NKG2C-BUV563 (clone 134591, BD Optibuild), anti-KIR2DL1/S1-APC (clone EB6B, Beckman Coulter), anti-KIR2DL1/S5-PE (clone 134211, R&D Systems), anti-KIR2DL2/L3/S2-BV711 (clone DX27, BD Optibuild), anti-KIR2DL3-AF488 (clone 180701, R&D Systems), anti-KIR3DL1-AF700 (clone DX9, BioLegend), anti-KIR3DL1/L2-PE-Vio770 (clone 5.133, Miltenyi) and anti-KIR2DS4-Biotin (clone JJC11.6, Miltenyi) with secondary Strepdavidin-BV421 (BioLegend). Cells were washed and then fixed with FluoroFix Buffer (BioLegend). Cells were analyzed by flow cytometry.
5
Measuring KIR2DL-Fc Binding to HLA-C
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HLA-C-721.221 cell lines and untransduced 721.221 were incubated with 25 μg/ml KIR2DL-Fc fusion protein for 15 min at 4°C and then washed and stained with a LIVE/DEAD fixable Near-IR Dead Cell staining kit (Invitrogen) and with a secondary F(ab)2 goat anti-human IgG-PE antibody (Invitrogen). After additional washing steps, the cells were fixed in 1x CellFIX (BD Bioscience) and the binding of the KIR2DL-Fc fusion protein was analyzed via flow cytometry. As a negative control, cells were only stained with anti-human IgG-PE without any KIR2DL-Fc fusion protein. HLA-C expression of all transfected and untransfected 721.221 cell lines was assessed by flow cytometry using the HLA-ABC antibody W6/32 (Supplementary Figure 1
6
Multiparametric Flow Cytometry of Tumor Immune Landscape
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One million cells isolated from tumor tissues were stained using the Live/Dead Fixable Near-IR Dead Cell staining kit (Invitrogen, Thermo Fisher Scientific), followed by cell fixation and permeabilization according to the manufacturer's (BD Pharmingen) protocol. For surface markers, cells were stained with appropriate antibodies in FACS buffer [1% FBS/BSA, 0.09% sodium azide, and 5 mmol/L EDTA (Sigma-Aldrich)] for 30 minutes at 4°C in the dark and washed twice in FACS buffer before acquisition. For IFNγ and IDO staining, BD Cytofix/Cytoperm (catalog no. 51-2090KZ) and BD Perm/Wash (catalog no. 51-2091KZ) buffer set were used as per manufacturer's instructions (BD Pharmingen). Data acquisition was performed on FACSCalibur or LSRII (BD Biosciences). Total numbers or frequency of CD3+, CD8+, CD8+E7+, E7+GB+, CD40L+, IFNγ+, CD4+, CD4+Foxp3+, CD11b+F4/80+, CD11c+, CD11b+Gr1+, CD80+, CD206+, B220+, and IDO+ cells were analyzed within the CD45+ hematopoietic cell population and represented on respective populations. For checking the expression of IDO, TC-1 and B16F10 cancer cell lines were stained with IDO antibody as described above. Results were analyzed with FlowJo (TreeStar) software.
7
Fluorescent Antibody-Based Immune Profiling
8
Comprehensive Immune Cell Phenotyping
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