The following conjugated antibodies were used for flow cytometric phenotyping and analysis: anti-CD34, anti-CD43, anti-KDR, anti-CD45, anti-CD7, anti-CD5, anti-CD4, anti-CD8, anti-CD3, antiTCRαβ, anti-CD56, anti-CD15, anti-CD14, and anti-CD235a purchased from BD Biosciences. All antibodies were used in a 1:30 dilution. Dead cells were excluded from analysis in all experiments by staining with DAPI. Flow cytometry analysis was conducted on an LSRII cytometer (BD Biosciences, Paris, France), a FACS CANTO (BD Biosciences, Paris, France), or a FACS CELESTA (BD Biosciences, Paris, France) and analyzed using FlowJo software (BD Biosciences, Ashland, OR, USA).
Anti cd5
Manufactured by BD
Sourced in United States
Anti-CD5 is a monoclonal antibody that binds to the CD5 antigen expressed on the surface of T cells and a subset of B cells. It is used in laboratory research applications to identify and study these cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd8, by BD (5 mentions)
Anti cd4, by BD (3 mentions)
Anti cd56, by BD (2 mentions)
Anti cd43, by BD (2 mentions)
Anti cd3, by BD (2 mentions)
Anti cd19, by BD (2 mentions)
Apc conjugated anti il 7rα, by Miltenyi Biotec (2 mentions)
Fitc conjugated anti t1 st2, by Miltenyi Biotec (2 mentions)
Biotin conjugated anti cd4, by BD (2 mentions)
Pe conjugated anti streptavidin, by BD (2 mentions)
Apc conjugated anti il 7rα, by BioLegend (2 mentions)
Biotin conjugated anti fcεri antibodies, by BioLegend (2 mentions)
Pecy7 conjugated c kit, by Thermo Fisher Scientific (2 mentions)
Anti ter119, by BD (2 mentions)
Anti gr 1, by BD (2 mentions)
Anti b220, by BD (2 mentions)
Anti nk1, by BD (2 mentions)
Apc cy7 conjugated anti cd25, by BD (2 mentions)
Anti cd11b, by BD (2 mentions)
Macsquant analyzer, by Miltenyi Biotec (2 mentions)
10 protocols using anti cd5
1
Flow Cytometric Phenotyping of Immune Cells
2
Comprehensive Immune Cell Profiling
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Freshly prepared PBMCs were used. Subpopulations of T cells, B cells, natural killer (NK) cells and antigen-presenting cells (APC) were characterized by surface staining with fluorescence labelled anti-CD3, anti-CD4, anti-CD5, anti-CD8, anti-CD14, anti-CD16, anti-CD19, anti-CD25, anti-CD27, anti-CD38, anti-CD45RA, anti-CD45RO, anti-CD56 and anti-HLA-DR (BD Bioscience); anti-BDCA1, anti-BDCA2, anti-BDCA3, anti-BDCA4 and anti-slan (Miltenyi Biotec). Negative controls included directly labeled or unlabeled isotype-matched irrelevant antibodies (BD Biosciences). Freshly prepared CSF cells were used directly for FACS analysis. Fluorescence-labeled antibodies for surface staining were used as follows: anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD16, anti-CD19, anti-CD25, anti-CD27, anti-CD45RA, anti-CD45RO, anti-CD56 and anti-HLA-DR (BD Bioscience); anti-BDCA1 and anti-slan (Miltenyi Biotec). All cells were measured on a LSR-Fortessa (BD Biosciences) and evaluated by FACS-Diva Software (BD Bioscience).
3
Multiparametric Flow Cytometry Analysis
4
Immunophenotyping of Murine Leukemia Cells
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Preparation of single-cell suspensions of lymphoid organs and lysis of red blood cells were performed according to standard procedures. Cells were (in)directly stained in flow cytometry buffer (PBS, supplemented with 0.25% BSA, 0.5 mM EDTA and 0.05% sodium azide) using the following fluorochrome or biotin-conjugated monoclonal antibodies or reagents: anti-B220 (RA3-6B2), anti-CD19 (ID3), anti-CD5 (53-7.3), anti-CD43 (R2/60), anti-CD23 (B3B4) all from eBioscience and anti-CD138 (281-2), anti-CD95 (Jo2), anti-IgD (11-26), anti-IgMb (AF6-78), anti-IgMa (DS-1), anti-Igλ (R26-46), anti-Igκ (187.1), anti-CD21 (7G6), all from BD biosciences, using conjugated streptavidin (eBioscience) as a second step for biotin-conjugated antibodies.
Leukemic cells (CD19+CD5+) were stained with fluorescein-labeled phosphatidylcholine (PtC) liposomes (DOPC/CHOL 55:45, Formumax Scientific Inc.) in flow cytometry buffer. Cells were co-stained with anti-CD19, anti-CD43, or anti-CD5 (BD Biosciences).
Leukemic cells (CD19+CD5+) were stained with fluorescein-labeled phosphatidylcholine (PtC) liposomes (DOPC/CHOL 55:45, Formumax Scientific Inc.) in flow cytometry buffer. Cells were co-stained with anti-CD19, anti-CD43, or anti-CD5 (BD Biosciences).
5
Calpain Inhibition in Inflammatory Response
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Sodium taurocholate was obtained from Sigma (St. Louis, USA). Emodin was purchased from Dalian Food and Drug Administration (Dalian, China). The calpain inhibitor (PD150606) was obtained from Alexis Biochemicals (San Diego, USA). The amylase, TNF-α, and IL-6 assay kits were obtained from Lengton Bioscience Co. (Shanghai, China). 4% paraformaldehyde was purchased from Solarbio (Beijing, China). Dextran T500 was obtained from Sigma (St. Louis, USA). Anti-CD 2, anti-CD 5, and anti-CD 45R were purchased from PharMingen (San Diego, USA). Anti-F4/80 was purchased from AbD Serotec (Oxford, UK). Anti-ICAM-1 was purchased from eBioscience (San Diego, USA). Goat anti-rabbit IgG microbeads were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The Fluo-3-AM assay kit was obtained from Dojindo Laboratories (Kyushu, Japan). Antibodies against calpain 1, caspase 12, caspase 3, β-tubulin, and goat anti rabbit IgG (H+L) secondary antibody were obtained from Abcam (Cambridge, UK). Calpain activity assay kit was obtained from Genmed (Boston, USA). The apoptosis detection kit (Annexin V-FITC/PI) was purchased from Solarbio (Beijing, China).
6
Immunophenotyping of RT-DLBCL cells
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To determine the immunophenotype of the RT-DLBCL cells, HPRT3, HPRT2, and HPRT1 cells were harvested from NSG mice. Cells were suspended in 100 µL of 0.5% BSA/PBS and stained with fluorophore-conjugated anti-CD19, anti-CD5, anti-CD10, anti-CD20, anti-CD23, anti-PD-1 or IgG-isotype controls (BD Biosciences, San Jose, CA). Percent expression of each cell surface marker is reported relative to the respective IgG isotype control.
7
Lung Innate Lymphoid Cell Type 2 Isolation
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The cells isolated from digested lungs were stained with biotin-conjugated antibody mixtures for lineage markers (CD4, CD5, CD8, CD11c, CD11b, CD19, NK1.1, Gr-1, TER119, FcεRI, and B220), Pacific blue-conjugated anti-Sca-1, PECy7-conjugated c-Kit (CD117), APC-conjugated anti-IL-7Rα (CD127), FITC conjugated anti-T1/ST2, APC-Cy7-conjugated anti-CD25, and PE conjugated anti-streptavidin, and analyzed using a MACSQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). Lin−Sca+c-Kit+IL-7R+CD25+ST2dim cells were identified as lung ILC2s [21 (link), 22 (link)]. The data were analyzed using FlowJo (TreeStar, Ashland, OR, USA). APC-Cy7-conjugated anti-CD25, Pacific blue-conjugated anti-Sca-1, biotin-conjugated anti-CD4, anti-CD5, anti-CD8, anti-CD11b, anti-NK1.1, anti-Gr-1, anti-TER119, and anti-B220, and PE-conjugated anti-streptavidin antibodies were obtained from BD Biosciences (Franklin Lakes, NJ, USA). FITC-conjugated anti-T1/ST2 was from MD Bioscience (St Paul, MN, USA). APC-conjugated anti-IL-7Rα and biotin-conjugated anti-FcεRI antibodies were from BioLegend (San Diego, CA, USA). PECy7-conjugated c-Kit was from eBioscience (La Jolla, CA, USA). Biotin-conjugated anti-CD11c and anti-CD19 were from TONBO Biosciences (San Diego, CA, USA).
8
Comprehensive Multiparametric T Cell Analysis
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Stained cells were analyzed using an LSRII system (BD Biosciences). Data were analyzed with the Diva software v.8 (BD Biosciences) and FlowJo v.10. Cell viability was evaluated using SYTOX Blue (Life Technologies). The following antibodies were used: anti-CD5 (BD Biosciences, catalog no. 550035, clone 53–7.3, DF 1:800), anti-CD4 (BD Biosciences, catalog no. 557956, clone RM4-5, DF 1:800), anti-CD8α (BD Biosciences, catalog no. 563046, clone 53–6.7, DF 1:400), anti-TCRβ (BD Biosciences, catalog no. 562839, clone H57-597, DF 1:200), anti-CD44 (BD Biosciences, catalog no. 560569, clone IM7, DF 1:800), anti-CD69 (BD Biosciences, catalog no. 553236, clone H1.2F3, DF 1:400), anti-CD6 (BD Biosciences, catalog no. 566426, clone J90-462, DF 1:800), anti-CD3ε (Biolegend, catalog no. B209683, clone 145-2C11, DF 1:200) and the anti-IFN-γ (Biolegend, catalog no. 505826, clone XMG1.2, DF 1:600).
9
Isolation and Characterization of Peritoneal B Cell Subsets
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Flow cytometric analysis and cell sorting were performed as previously described (36 (link)). Briefly, peritoneal washout cells were obtained from five different mice of each DH-altered strain. The following mAbs were used to isolate PerC B cells into the B-1a, B-1b, and B-2 subpopulations: anti-B220 (RA 3.6B2) (BD Pharmingen) (Southern Biotechnology, Birmingham, AL, USA), anti-Mac-1 (BD Pharmingen, San Diego, CA, USA) and anti-CD5 (BD Pharmingen, San Diego, CA, USA). PerC B-1a cells were sorted as B220loCD5+, B-1b as B220loCD5−Mac-1lo/+, and PerC B-2 cells were sorted as B220hiCD5−Mac-1−. A MoFlo instrument (Dako) was used for cell sorting.
10
Identification and Analysis of Lung ILC2s
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Analyses of ILC2s. The cells isolated from digested lungs were stained with biotin-conjugated antibody mixtures for lineage markers (CD4, CD5, CD8, CD11c, CD11b, CD19, NK1.1, Gr-1, TER119, FcεRI, and B220), Paci c blue-conjugated anti-Sca-1, PECy7-conjugated c-Kit (CD117), APC-conjugated anti-IL-7Rα (CD127), FITC conjugated anti-T1/ST2, APC-Cy7-conjugated anti-CD25, and PE conjugated antistreptavidin, and analyzed using a MACSQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany).
Lin -Sca + c-Kit + IL-7R + CD25 + ST2 dim cells were identi ed as lung ILC2s [21] [22] . The data were analyzed using FlowJo (TreeStar, Ashland, OR, USA). APC-Cy7-conjugated anti-CD25, Paci c blue-conjugated anti-Sca-1, biotin-conjugated anti-CD4, anti-CD5, anti-CD8, anti-CD11b, anti-NK1.1, anti-Gr-1, anti-TER119, and anti-B220, and PE-conjugated anti-streptavidin antibodies were obtained from BD Biosciences (Franklin Lakes, NJ, USA). FITC-conjugated anti-T1/ST2 was from MD Bioscience (St Paul, MN, USA). APCconjugated anti-IL-7Rα and biotin-conjugated anti-FcεRI antibodies were from BioLegend (San Diego, CA, USA). PECy7-conjugated c-Kit was from eBioscience (La Jolla, CA, USA). Biotin-conjugated anti-CD11c and anti-CD19 were from TONBO Biosciences (San Diego, CA, USA).
Lin -Sca + c-Kit + IL-7R + CD25 + ST2 dim cells were identi ed as lung ILC2s [21] [22] . The data were analyzed using FlowJo (TreeStar, Ashland, OR, USA). APC-Cy7-conjugated anti-CD25, Paci c blue-conjugated anti-Sca-1, biotin-conjugated anti-CD4, anti-CD5, anti-CD8, anti-CD11b, anti-NK1.1, anti-Gr-1, anti-TER119, and anti-B220, and PE-conjugated anti-streptavidin antibodies were obtained from BD Biosciences (Franklin Lakes, NJ, USA). FITC-conjugated anti-T1/ST2 was from MD Bioscience (St Paul, MN, USA). APCconjugated anti-IL-7Rα and biotin-conjugated anti-FcεRI antibodies were from BioLegend (San Diego, CA, USA). PECy7-conjugated c-Kit was from eBioscience (La Jolla, CA, USA). Biotin-conjugated anti-CD11c and anti-CD19 were from TONBO Biosciences (San Diego, CA, USA).
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