To prepare samples for scRNA-seq analysis, bone marrow, lymph node and peripheral blood cells isolated from C57BL/6 J female mice were stained with Live/Dead dye (Fixable Viability Stain 510, cat# 564406, BD Pharmingen) and lymphocytes were isolated using a BD FACSAria™ III Sorter (Fig. 1a ). Cells were manually counted by Trypan blue (Thermo, T10282) and AO-PI (LUNA, D23001) after each centrifugation and resuspension. Single cells were processed using Chromium Controller (10x Genomics) according to the manufacturer’s protocol. By using Chromium Next GEM Single Cell 5’ Kit v2 (10x Genomics, 1000263) and Chromium Next GEM Chip K Single Cell Kit (10x Genomics, 1000287), we performed single cell TCR/BCR-seq and 5’ gene expression profiling. The cell suspension was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer’s protocol. Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs. Cell-barcoded 5’ gene expression libraries and V(D)J enriched TCR/BCR libraries were sequenced on an Illumina NovaSeq6000 system by Shanghai Biochip Co., Ltd., Shanghai, China.
Live dead dye
Manufactured by BD
Sourced in United States
The Live/Dead dye is a fluorescent staining reagent used to assess cell viability. It provides a rapid and straightforward method to distinguish live and dead cells in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Tumor disassociation kit, by Miltenyi Biotec (2 mentions)
Facscalibur, by BD (2 mentions)
Anti cd4 fitc, by BD (2 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Fixable viability stain 510, by BD (1 mentions)
Chromium controller, by 10x Genomics (1 mentions)
Novaseq 6000 system, by Illumina (1 mentions)
Chromium next gem single cell 5 kit v2, by 10x Genomics (1 mentions)
Chromium next gem chip k single cell kit, by 10x Genomics (1 mentions)
Chromium single cell controller, by 10x Genomics (1 mentions)
Facsaria 3 sorter, by BD (1 mentions)
Anti granzyme b pe, by BD (1 mentions)
Anti ifnγ pe cf594, by BD (1 mentions)
Anti foxp3 percp, by BD (1 mentions)
Anti ki 67 apc, by BD (1 mentions)
Anti cd8 pe cy7, by BD (1 mentions)
Golgistop, by Thermo Fisher Scientific (1 mentions)
Monesin, by Thermo Fisher Scientific (1 mentions)
Anti cd11b pe cy7, by BD (1 mentions)
Anti ly6c apc, by BD (1 mentions)
4 protocols using live dead dye
1
Single-Cell Profiling of Murine Immune Cells
2
Tumor-Infiltrating Immune Cell Profiling
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Tumor tissue was harvested and cut into small fragments followed by digestion with tumor disassociation kit for 30 min (Miltenyi Biotec, USA), and then filtered by 70 µm cell strainers. Mononuclear cells were enriched by percoll gradient centrifuging of the single cell suspension. Cells were washed with PBS and stained with Live/Dead dye, anti-CD4 FITC, anti-CD8 PE-Cy7, anti-Ki67 APC, anti-IFNγ PE-CF594, anti-FoxP3 Percp and anti-granzyme B PE antibodies, along with appropriate isotype controls (all from BD) for flow cytometry analysis (BD FACSCalibur).
3
Phenotyping and Sorting of G-MDSC and M-MDSC
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G-MDSC and M-MDSC were phenotyped using flow cytometry. Tumor was cut into small fragments followed by digestion for 30 min with tumor disassociation kit (Miltenyi Biotec, USA), and then filtered by 70 µm cell strainers. The percoll gradient was used to enrich mononuclear cells. After washing with PBS, cells were stimulated with PMA/ionomyocin in the presence of Golgi stop and Monesin (eBioscience, San Diego, CA, USA) for 4 h. Cells were washed with PBS and then stained with Live/Dead dye, anti-CD11b PE-Cy7, anti-Ly6G AF700, anti-Ly6C APC, anti-CD4 FITC and anti-CD8 Percp antibodies (all from BD). After fixation-permeabilization, cells were stained with anti-IFNγ PE-CF594 antibody, along with appropriate isotype controls (all from BD) for flow cytometry analysis (BD FACSCalibur). G-MDSC was defined as CD11b+Ly6G+Ly6C- and M-MDSC was defined as CD11b+Ly6C+Ly6G-. MDSC subsets were sorted with corresponding beads from the tumor lysates for western blot assay.
4
Phenotypic Analysis of Immune Cells
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For phenotypic analysis, single-cell suspensions were prepared from spleens, inguinal lymph nodes, submandibular lymph nodes, tertiary lymphoid structures (TLS) and the blood of WT, BBimfl/fl, Bim−/−, Btk−/− and BBimfl/fl × Btk−/− mice. Cells were stained with fluorescently labelled antibodies in various combinations to identify; B cells and subpopulations including T1, MZ, pMZ, An1, CD21lo and, CD4 and CD8 T cells and their subpopulations including T follicular cells, as defined below. Antibodies, fluorochrome labelling and sources are detailed in Table 1
Intracellular cytokine staining was carried out by first staining cell surface markers in PBS with 2% serum after incubation with FcR-block (CD16), washed and stained with antibodies to various cytokines (IFN-γ, IFN-α, IL-6, IL-10 and TNF-α) using the BD Biosciences fix/perm kit. Dead cells and doublets were excluded from the FCM analysis by Live/Dead dye (BD Biosciences) and SSC-W/SCC-H and FCS-W/FSC-H gating protocols. Dead cells and cells undergoing apoptosis were detected by staining with AnnexinV and 7AAD. All flow cytometry data was acquired on a BD LSR II flow cytometer and analyzed using the FlowJo software package (Tree Star).
Intracellular cytokine staining was carried out by first staining cell surface markers in PBS with 2% serum after incubation with FcR-block (CD16), washed and stained with antibodies to various cytokines (IFN-γ, IFN-α, IL-6, IL-10 and TNF-α) using the BD Biosciences fix/perm kit. Dead cells and doublets were excluded from the FCM analysis by Live/Dead dye (BD Biosciences) and SSC-W/SCC-H and FCS-W/FSC-H gating protocols. Dead cells and cells undergoing apoptosis were detected by staining with AnnexinV and 7AAD. All flow cytometry data was acquired on a BD LSR II flow cytometer and analyzed using the FlowJo software package (Tree Star).
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