Blood was taken routinely from anesthetized mice (retrobulbar venous plexus). Spleen and tumor tissues were dissociated. Single cells were stained with a panel of conjugated monoclonal antibodies (mAb, 0.125 μg to 1.5 μg each). Zombie NIR™ Fixable Viability Kit by Biolegend (San Diego, United States) staining was performed following the protocol Zombie NIR™ Fixable Viability Kit by Biolegend, extracellular staining was performed following the protocol BD Horizon Briliant Stain Buffer (BD Bioscience), followed by lysis and intracellular staining using the protocol of True-Nuclear™ Transcription Factor Buffer Set by Biolegend. Measurements were performed on a spectral flow cytometer (Cytek™ Aurora). For extracellular stainings Gr1 Alexa Fluor700, CD8 FITC, CD4 APC Fire, CD11b BV570, PD-L1 BV421, NK1.1 BV605, CD19 Spark Blue (Biolegend), CD25 PerCP-eFluor710 (Thermofisher), CD83 BV750, PD-1 BV650 (BD Bioscience) and for intracellular stainings CTLA-4 PE/Cy7, CD3 PerCP, and Foxp3 Alexa Fluor 647 (Biolegend) were used. Data were analyzed using SpectroFlow™ Version 2.2.0.3. and FlowJo™ Version 10.6.1.
Zombie nir fixable viability kit
Manufactured by BioLegend
Sourced in United States, United Kingdom
The Zombie NIR Fixable Viability Kit is a fluorescent dye-based kit used to identify and quantify dead cells in a cell population. The kit utilizes a near-infrared (NIR) fluorescent dye that stains dead cells, allowing for their detection and discrimination from live cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (15 mentions)
Facscanto 2, by BD (11 mentions)
Lsrfortessa x 20, by BD (9 mentions)
Lsrfortessa flow cytometer, by BD (9 mentions)
Facscanto 2 flow cytometer, by BD (7 mentions)
Lsr 2, by BD (6 mentions)
Cytofix cytoperm, by BD (6 mentions)
Cell activation cocktail, by BioLegend (5 mentions)
Trustain fcx, by BioLegend (5 mentions)
True nuclear transcription factor buffer set, by BioLegend (5 mentions)
Cytoflex, by Beckman Coulter (5 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (5 mentions)
Facsverse, by BD (5 mentions)
Facsaria 2, by BD (5 mentions)
Anti cd45, by BioLegend (5 mentions)
Cell staining buffer, by BioLegend (4 mentions)
Facsaria 3, by BD (4 mentions)
Lsrfortessa x 20 flow cytometer, by BD (4 mentions)
Facsdiva software, by BD (4 mentions)
Lsrii system, by BD (3 mentions)
199 protocols using zombie nir fixable viability kit
1
Multiparametric Flow Cytometry of Murine Immune Cells
2
Evaluation of BMDC Maturation and T Cell Proliferation
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To test BMDC maturation, cells were stained with live-dead marker (Zombie-NIR or Zombie-Aqua Fixable Viability Kit, Biolegend) in PBS before staining in incubation buffer (1% FBS in PBS) with FCR (FC receptor) blocking antibody (Biolegend, clone 93) and antibodies against CD11c, MHC II, CD80, CD86, CD40 and ICAM1 (Supplementary file 2 ). Cells were acquired on a BD LSRFortessa. Data were analysed in FlowJo (v10) gating on single, live cells. Mature BMDCs were consistently >90% CD11c+ and MHC II+ (Figure 7—figure supplement 1b ). To measure CD80 and ICAM-1 expression on activated T cells, T cells were stained with live-dead marker (Zombie-NIR Fixable Viability Kit, Biolegend) and antibodies against CD80 and ICAM1 in the same way.
To test the impact of inhibiting the MEK and mTOR pathways on cell proliferation (Figure 3—figure supplement 3g ), cells were stained with eBioscience Cell Proliferation Dye eFluor-450 (ThermoFisher), pre-treated for 2 hr with MEK162 (1 µM and 5 µM), rapamycin (200 nM) or combined MEK162 (1 µM or 5 µM) and rapamycin (200 nM), and stimulated with 1 µM N4 or NP68 peptides for 2 days. Cells were then stained with live-dead marker (Zombie-NIR Fixable Viability Kit, Biolegend) and acquired on a BD LSRFortessa. Data were analysed in FlowJo (v10) gating on live, single cells.
To test the impact of inhibiting the MEK and mTOR pathways on cell proliferation (
3
Nanotrap Binding Kinetics Characterization
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For Nanotrap-virus binding, SARS-CoV-2 spike pseudotyped lentivirus was labeled with lipophilic dye DiO75 (link) for 20 min, fixed with 4% PFA, then washed three times with PBS in an Amicon Ultra-centrifugal tube (100 kDa). DiD-loaded Nanotraps were prepared and incubated with the DiO-labeled pseudotyped lentivirus for various times. DiD+DiO+ double-positive events were gated, and the mean fluorescence intensity of DiO was recorded by flow cytometry until saturation was achieved.
For Nanotrap-macrophage binding, macrophages were prepared as described above and incubated with DiO-loaded Nanotraps for varying times until saturation was achieved. The cells were harvested and washed three times with PBS at 300 × g for 5 min, and stained with a Zombie NIR Fixable Viability Kit (BioLegend) on ice for 10 min. The cells were then washed with FACS buffer two times and resuspended in 200 μL of FACS buffer. Flow cytometry was carried on a BD LSRFortessa Flow Cytometer. Live and single cells were gated, and the DiO fluorescence channel was used to indicate the phagocytosis efficiency at various time points.
For Nanotrap-macrophage binding, macrophages were prepared as described above and incubated with DiO-loaded Nanotraps for varying times until saturation was achieved. The cells were harvested and washed three times with PBS at 300 × g for 5 min, and stained with a Zombie NIR Fixable Viability Kit (BioLegend) on ice for 10 min. The cells were then washed with FACS buffer two times and resuspended in 200 μL of FACS buffer. Flow cytometry was carried on a BD LSRFortessa Flow Cytometer. Live and single cells were gated, and the DiO fluorescence channel was used to indicate the phagocytosis efficiency at various time points.
4
Multicolor Flow Cytometry Staining
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Cells were stained with Zombie NIR Fixable Viability Kit (Biolegend) or Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific), followed by the staining with fluorescent-labeled antibodies for 15 min. The cells were then washed and resuspended in MACS buffer (0.5% bovine serum albumin (Sigma–Aldrich), 2 mM EDTA and 1× DPBS). For intracellular staining, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Flow cytometric data were collected using FACSVerse or LSRFortessa X-20 (BD Biosciences) or SH800Z (Sony). Collected data were analyzed with FlowJo (BD Biosciences).
5
Comprehensive Immunophenotyping of Immune Cells
6
Analysis of SARS-CoV-2 Spike Protein-Specific T Cells
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Cryopreserved PBMCs were thawed and stimulated with 1 μg/mL mega-pool Spike peptides or equivalent amount of DMSO for 24 h at 37°C. Cells were then washed in phosphate buffered saline and stained with Zombie NIR Fixable Viability Kit (Biolegend) to exclude dead cells in the analysis. The cells were next washed in FACS buffer with 2mM EDTA and surface markers were stained with the surface markers, anti-CD3, anti-CD4, anti-CD8, anti-CD25, anti-CD69, anti-CD134 (OX40) and anti-CD137 (4-1BB), diluted in FACS buffer for 30 min on ice. After two more washes in FACS buffer, cells were resuspended in PBS prior to acquisition with Beckman Coulter CytoFLEX S analyser.
7
Single-cell Viability Analysis by Flow
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Single-cell suspensions were counted and resuspended in PBS at a concentration of 107 cells/mL. One hundred microliters of each sample were stained with Zombie NIR Fixable Viability kit (BioLegend) following the manufacturer’s instructions. All flow cytometry analyses were performed using a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar).
8
IL-22 Stimulation of Monocytes
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For analysis of the DARC+ subset, human THP-1 cells, primary PBMCs isolated from human blood, or bone marrow cells from 8-week-old female C57BL/6J mice were stimulated for 24 h with 20 or 40 ng/mL of IL-22. CD14+ monocytes (for human), monocytes (CD11b+), and macrophages (F4/80+) (for mouse) were then sorted for expression analysis. Zombie NIR™ Fixable Viability kit (Biolegend) was used to exclude cell debris and dead cells. Sorting was carried out on a BD FACSCanto II (BD Biosciences) and >90% of the target population was obtained.
9
Characterizing Immune Cell Populations in Lung Tissue
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Inflammatory cells in lung tissue were analyzed to identify eosinophils (CD45+CD11b+Ly6C-SiglecF+), Th17 cells (CD45+CD4+IL-17A+), regulatory T (Treg) cells (CD45+CD4+CD25+Foxp3+) by flow cytometry as previously described (Zhao et al., 2013 (link); Cossarizza et al., 2017 (link); Dong et al., 2018 (link)). Briefly, the excised right lung was digested by using lung dissociation kits (MiltenyiBiotec Technology & Trading Co. Ltd, Shanghai, China) to prepare single cell suspensions following the instructions. And then the cells were stained with the monoclonal anti-murine fluorochrome-conjugated Abs and detected by an Attune NxT instrument (Life Technology). Besides, the antibodies used were bought from BioLegend, including eflour506-labeled anti-CD45, Zombie NIR™ Fixable Viability Kit, Brilliant Violet 421™-labeled anti-CD11b, APC-labeled anti-Siglec-F, PE-labeled anti-Ly-6C, APC-labeled anti-CD25, FITC-labeled anti-CD4, PE-labeled anti-IL-17. PE-labeled anti-Foxp3 was purchased from eBioscience.
10
Surface Marker Profiling of Cells
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For surface staining, cells were kept at 4°C. Foxp3/Transcription Factor Staining Buffer Kit was used for intracellular staining following the manufacturer’s instructions (TNB-0607, KIT). Antibodies used were as follows: AF480-Rabbit-α–vWF (ab195028, Abcam), PeCy7-Mouse-α–PECAM (563651, BD), APC-Mouse-α–β1-Integrin (559883, BD), AF 647-Mouse-α–β3-Integrin (336407, Biolegends), PE-Mouse-α–DAF (555694, BD), and α–PCDH1 mAb 3305 (2)/AF488-α–Human (A-11013, Invitrogen). Zombie NIR Fixable Viability Kit (BioLegend) was used to assess live/dead status of the cells.
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