All samples were run on the LSR II and data were analysed using FlowJo v10.6.1 (BD Biosciences, San Jose, CA). Briefly cells were stained on ice for 15–20 min with the viability dye Ghost Dye BV510 (Tonbo Bioscience, cat# 13–0870) and the following antibodies: CD45 (Biolegend, cat#103134), CD8 (Tonbo Bioscience, cat# 35–0081), CD44 (Tonbo Bioscience, cat# 65–0441), CD4 (Biolegend, cat# 100456), CD28 (Biolegend, cat# 102104), CD62L (Tonbo Bioscience, cat#60‐0621), CD3 (Tonbo Bioscience, cat#80‐0032), CD69 (Biolegend, cat#104530), CCR7 (Biolegend, cat# 120124), CD25 (Biolegend, cat#102012), Foxp3 (Invitrogen, cat#12‐5773‐82), and CD103 (Biolegend, cat# 121426). Cells were subsequently washed and fixed using the Foxp3 Transcription Factor staining buffer set (ThermoFisher Scientific, cat#00‐5523‐00). The cells used for scRNAseq were depleted of EpCAM+ cells using the EasySep Mouse Streptavidin RapidSpheres Isolation Kit (Stem Cell, cat#19860) and then sorted using the MoFlo sorting system based on the expression of CD45 (Biolegend, cat#103112) and viability (Ghost Dye BV510, Tonbo Bioscience, cat# 13–0870).
Ghost dye bv510
Manufactured by Cytek Biosciences
Sourced in United States
The Ghost Dye-BV510 is a fluorescent dye designed for use in flow cytometry applications. It is used for the identification and enumeration of viable cells within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Software, by FlowJo (3 mentions)
Anti mouse cd16 cd32 fc blocked, by BD (2 mentions)
Cd8 pe cy5, by Cytek Biosciences (2 mentions)
Cd44 perp cy5, by Cytek Biosciences (2 mentions)
Cd44 pe cy7, by Cytek Biosciences (2 mentions)
Cxcr3 bv650, by Cytek Biosciences (2 mentions)
Lsrfortessa x 20, by BD (2 mentions)
Easysep mouse streptavidin rapidspheres isolation kit, by STEMCELL (1 mentions)
Foxp3, by Thermo Fisher Scientific (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Cd103, by BioLegend (1 mentions)
Fc block 2.4g2, by BioXCell (1 mentions)
Ma900 flow cytometer, by Sony (1 mentions)
Anti cd16 32, by BioLegend (1 mentions)
Lsrfortessa x 20 flow cytometer, by BD (1 mentions)
Cd8 pecy7, by Cytek Biosciences (1 mentions)
Cd11b apc cy7, by Cytek Biosciences (1 mentions)
7 protocols using ghost dye bv510
1
Comprehensive Immune Cell Profiling
2
DNFB-induced Flank Skin Leukocyte Isolation
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We used DNFB-challenged flank tissue rather than labiar skin or vaginal canal samples in order to obtain enough cells for analysis. To prepare flank tissue samples, ND4 female Swiss mice were shaved on the back (15 mm × 15 mm) and both flanks (15 mm × 15 mm) 1 day prior to sensitization. All mice were sensitized with a topical application of 25 µL of 0.5% DNFB (Sigma-Aldrich, St. Louis, MO, USA; #D1529-10ML) dissolved in 0.9% saline on the shaved back. Four days after sensitization, mice were challenged on both shaved flanks with 25 µL (50 µL total) of 0.3% DNFB dissolved in 0.9% saline or 0.9% saline alone daily for 10 consecutive days. Leukocytes were isolated from flank skin samples from CO2-euthanized mice as previously described [51 (link)] 1 day after the cessation of challenges. Cells were washed, blocked for five minutes with 50 μL of supernatant from a 2.4G2 hybridoma cell line (BioXCell, West Lebanon, NH, USA), stained with fluorochrome-conjugated monoclonal antibodies (Table 2 ) at 1:100 dilutions for 30 min at 4 °C. and analyzed using an LSR Fortessa X-20 (Becton Dickinson, Franklin Lakes, NJ, USA) flow cytometer. Acquired data were analyzed using FlowJo software (Version 10, FlowJo LLC, Ashland, OR, USA). Dead cells were excluded using Ghost Dye-BV510 (Tonbo Biosciences, San Diego, CA, USA).
3
Immune Cell Profiling in Influenza Infection
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The lungs were aseptically collected from mock- and IAV-infected mice, and single cells were prepared after collagenase digestion of lungs, as previously described (52 (link)). One million cells per sample were stained with Ghost Dye BV510 (Tonbo Biosciences, San Diego, CA) and anti-mouse CD16/CD32 (Fc-blocked) (BD Biosciences, San Jose, CA) at 4°C for 30 min. For cell surface staining, cells were incubated for 30 min at room temperature with anti-mouse CD3–allophycocyanin (APC)–Cyanine 7 (Cy7), CD3–Alexa Fluor 488, CD4–Brilliant Violet 786, CD8–phycoerythrin (PE)–Cy7 (Tonbo Biosciences), CD8-PE-Cy5, CD44-PerP-Cy5.5, CD44-PE-Cy7, CD62L–fluorescein isothiocyanate (FITC), CXCR3-BV650, and CXCR3-PE-Dazzle. For monocytes, cells were stained with Cd11b APC-Cy7, Ly6C-BV711, Ly6G-FITC, and CCR2-PE-Cy7. All the antibodies were purchased from BioLegend unless specified.
4
Immunophenotyping of Iliac Lymph Nodes
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Harvested iliac lymph nodes were passed through a 70 μm strainer, washed with staining buffer (2% fetal bovine serum in 1× PBS; Serum Source International, Charlotte, NC, USA) and stained with antibodies for immunophenotyping (1:100 dilution; Table 1 ) in the presence of Fc block (2.4G2; BioXCell) for 30 min on ice, and analyzed on a BD LSR Fortessa X-20 (Becton Dickinson, Franklin Lakes, NJ, USA). Acquired data were analyzed using FlowJo software (Version 10, FlowJo LLC, Ashland, OR, USA). Dead cells were excluded using Ghost Dye-BV510 (Tonbo Biosciences, San Diego, CA, USA).
5
Intracellular Cytokine Staining of Lung T Cells
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For intracellular cytokine staining, 1 million lung single cells were Fc-blocked with anti-CD16/32 and LIVE/DEAD-stained with Ghost Dye BV510 (Tonbo Biosciences) and surface-stained as described above. The cells were fixed, permeabilized with CytoFix/CytoPerm, and stained with anti-mouse GzmB-conjugated BV421 and APC-conjugated perforin. The perforin and GzmB expression represent the response from bulk CD8+ T cells. For IFN-γ detection (IFN-γ), lung single cells prepared from mock- and IAV-infected mice were stimulated with 10 μM IAV peptide NP366–374 for 5 hours with brefeldin A, in RPMI medium containing 10% fetal bovine serum (FBS) and supplemented with antibiotics, before intracellular staining with anti-mouse IFN-γ–PE–Dazzle 594 (26 (link)). A BD FACSymphony or SONY MA900 flow cytometer was used to acquire 100,000 events, and data were analyzed using FlowJo (Tree Star Inc.). The list of the antibodies used in this study including their clone and catalog number is provided in the Supplementary Materials (table S1).
6
Analyzing Immune Cell Dynamics in Sensitized Mice
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For inguinal lymph node analyses, flank-sensitized mice were sensitized on the back and challenged on the labia. For all other flow cytometric-based experiments, mice were sensitized and as described above. Draining lymph node (dLN) cells were isolated by crushing dLNs through a 70μm strainer. For skin cell isolation, leukocytes were isolated from flank skin samples as previously described [23 (link)]. Cells were washed, blocked with 1:100 anti-CD16/32 (Biolegend, San Diego, CA, USA), stained with fluorochrome-conjugated monoclonal antibodies (anti-mouse CD45.1, CD45.2, CD3, CD4, CD44, CD19; Thermo Fisher; CD3; BD Biosciences; CD19, CD25; Biolegend; CD8a, CD4, CD103, CD117 (c-Kit), FcεR1; Tonbo Biosciences, San Diego, CA, USA) at 1:100 dilutions for 30 minutes and analyzed on a BD LSR Fortessa X-20 flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Dead cells were stained with Ghost Dye-BV510 (Tonbo Biosciences, San Diego, CA, USA) and excluded from analyses. Data were analyzed using FlowJo Software (Version X, FlowJo LLC, Ashland, OR, USA; gating strategy is outlined in S1 Fig ). Results were quantified as cell counts or mean ± SEM. Cell counts for each marker were calculated with their respective frequency of live cells and total cell count.
7
Flow Cytometric Analysis of Lung Immune Cells
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The lungs were aseptically collected from mock and IAV-infected mice, and single cells were prepared after collagenase digestion of lungs, as previously described (PMC7336542). One million cells per sample were stained with Ghost Dye-BV510 (Tonbo Biosciences, San Diego, CA) and anti-mouse CD16/CD32 (FC blocked) (BD Biosciences, San Jose, CA) for 30 min at 4°C for 30 min. For cell surface staining, cells were incubated for 30 min at room temperature with anti-mouse CD3-APC-Cy7, CD3-AF488, CD4-BV786, CD8-PE-Cy7(Tonbo), CD8-PE-Cy5, CD44 PerP-Cy5.5, CD44 PE-Cy7, CD62L-FITC, CXCR3-BV-650, CXCR3-PE-Dazzle; For monocytes, cells were stained with Cd11b APC/Cy7, Ly6C BV711, Ly6G FITC and CCR2 PE/Cy7. All the antibodies were purchased from Biolegend unless specified.
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