The single cell suspension obtained as described above was stained for cell sorting with a cocktail of biotinylated antibodies for lineage markers, anti-CD2, -CD3, -CD16, -CD64 (BD Biosciences; cat# 555325, 555338, 555405, and 555526), -CD31 (Invitrogen; cat# MHCD3115), -CD45 and -CD140b (BioLegend; cat# 304003 and 323604) to specifically remove hematopoietic, endothelial, and leukocyte lineage cells. Cells were stained for 20 min at room temperature in PBS with 1% BSA, followed by washing in PBS with 1% BSA. Biotinylated primary antibodies were revealed with an anti-human secondary antibody labeled with streptavidin-Pacific Blue conjugate (Invitrogen; cat# S11222). The lineage-positive and negative cell populations were sorted using a FACSAria II cell sorter (BD Biosciences). The lineage-positive population was discarded. The lineage-negative cell population was incubated simultaneously with three human-specific primary antibodies (anti CD-10 labeled with Phycoerythrin (PE) coupled to Cy7 (BD Biosciences; cat# 341092), anti-MUC1 labeled with FITC (BD Biosciences; cat# 559774) and anti-CD73 labeled with PE (phycoerythrin) (BD Biosciences; cat# 550257)) for 20 min at room temperature in PBS with 1% BSA, followed by washing in PBS with 1% BSA. Cells were then sorted as described above.
Anti cd2
Manufactured by BD
Sourced in United States
Anti-CD2 is a laboratory reagent used for the detection and analysis of CD2 (cluster of differentiation 2) expression on the surface of cells. CD2 is a cell surface glycoprotein expressed on T cells and natural killer cells, and it plays a role in cell adhesion and signal transduction. The Anti-CD2 reagent can be used in flow cytometry and other immunoassays to identify and characterize CD2-positive cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 2 cell sorter, by BD (2 mentions)
Fluorescein isothiocyanate (fitc), by BD (2 mentions)
Cd45 and cd140b, by BioLegend (2 mentions)
Streptavidin pacific blue conjugate, by Thermo Fisher Scientific (2 mentions)
Igg1 isotype control, by BioLegend (2 mentions)
Anti cd107a antibody, by BioLegend (2 mentions)
Anti nkg2d, by BioLegend (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Anti cd226, by BioLegend (2 mentions)
Goat anti rabbit igg h l secondary antibody, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
Sodium taurocholate, by Merck Group (1 mentions)
Anti f4 80, by Bio-Rad (1 mentions)
Anti icam 1, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg microbeads, by Miltenyi Biotec (1 mentions)
Dextran t500, by Merck Group (1 mentions)
Annexin 5 fitc pi, by Solarbio (1 mentions)
Calpain activity assay kit, by Genmed Scientifics (1 mentions)
Anti cd5, by BD (1 mentions)
Anti cd45r, by BD (1 mentions)
7 protocols using anti cd2
1
Isolation of Epithelial Progenitor Cells
2
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).
3
Lineage-Negative Cell Sorting for Tissue-Specific Isolation
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The single-cell suspension obtained as described above was stained for cell sorting with a cocktail of biotinylated antibodies for lineage markers, anti-CD2, -CD3, -CD16, -CD64 (BD Biosciences; cat no. 555325, 555338, 555405 and 555526), −CD31 (Invitrogen; cat no. MHCD3115), −CD45 and −CD140b (BioLegend; cat no. 304003 and 323604) to specifically remove hematopoietic, endothelial and leukocyte lineage cells. Cells were stained for 20 min at room temperature in PBS with 1% BSA, followed by washing in PBS with 1% BSA. Biotinylated primary antibodies were revealed with an anti-human secondary antibody labelled with streptavidin-Pacific Blue conjugate (Invitrogen; cat no. S11222). The lineage-positive and negative cell populations were sorted using a FACSAria II cell sorter (BD Biosciences). The lineage-positive population was discarded. The lineage-negative cell population was incubated simultaneously with three human-specific primary antibodies (anti CD10 labelled with Phycoerythrin (PE) coupled to Cy7 (BD Biosciences; cat no. 341092), anti-MUC1 labelled with fluorescein isothiocyanate (BD Biosciences; cat no. 559774) and anti-CD73 labelled with PE (BD Biosciences; cat no. 550257) for 20 min at room temperature in PBS with 1% BSA, followed by washing in PBS with 1% BSA. Cells were then sorted as described above.
4
Isolation of Large Pre-B Cells
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For isolation of large pre-B cells, also described as early pre-B cells that express pre-BCR and are highly proliferative (for review, see Hardy and Hayakawa 2001 (link)), BM cell preparations were depleted of cells binding to anti-Ter119, anti-Mac-1, anti-Gr-1, anti-IgM, anti-CD3, anti-CD8α, anti-TCRβ, and anti-DX5 (Supplemental Table S1 ) by removal with magnetic beads conjugated to BioMag goat anti-rat IgG (Qiagen, 310107). The cells remaining after depletion were labeled with fluorochrome-conjugated monoclonal antibodies to B-cell markers (anti-CD19 [eBiosciences, 25-0193], anti-CD43 [BD, 553270], anti-BP1 [Ebiosciences, 11-5995], anti-CD25 [BD, 553075], and anti-CD2 [BD, 553112]) and were used for flow cytometry. Large pre-B cells from wild-type, IkE5fl/fl CD2-Cre, or IkE5fl/flERT2-Cre were sorted as CD19+CD43+BP1+ using a MoFlo-Legacy (Cytomation) cell sorter.
5
NK Cell-Mediated Cytotoxicity Assay
6
Isolation of Proarteriogenic Monocytes
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We used TIE2 as a marker of proarteriogenic Mo/MΦs, and cells were isolated according to previously published methodology (Supplemental Figure 4 ) (15 (link)). Briefly, peripheral blood mononuclear cells were isolated from 100 mL of venous blood by density centrifugation (at 400g, spun for 30 minutes at 4°C) using Ficoll-Paque (GE Healthcare). Whole-population Mo were magnetically isolated using anti-CD14 microbeads (Miltenyi Biotec) followed by FACS for proarteriogenic Mo/MΦs (Aria II; BD Biosciences). The following antibodies were used for cell sorting: human anti-TIE2 (R&D Systems), anti-CD14, anti-CD2, anti-CD3, anti-CD19 and anti-CD56 antibodies (BD Biosciences) (Supplemental Table 10 ).
7
NK Cell Cytotoxicity Assay with HNSCC Targets
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NK cells were stimulated at indicated time points with HNSCC targets for 6 hours in presence of 1 ng/mL rhIL15. Effector to target (E:T) ratio was 5:1, unless otherwise indicated, with anti-CD107a antibody (Biolegend; RRID:AB_1227509) for 6 hours, with Golgi Plug/Stop present in the last 5 hours. When indicated, NK cells were pre-incubated, IgG1 Isotype control (5μg/mL; BioLegend; RRID:AB_2801451), anti-NKG2D (5μg /mL; BioLegend; RRID:AB_2810480), anti-CD2 (5 μg/mL; BD-Biosciences; RRID:AB_395731) or anti-CD226 (5μg/mL; BioLegend; RRID:AB_1279155) blocking antibodies 30 minutes before incubation with tumor targets. In Cetuximab experiments, tumor cells were pre-incubated with anti-EGFR antibody cetuximab (10μg/mL; Lily) prior to incubation with NK cells. Cells were stained for flow cytometry analysis as described previously. 19 Degranulation (CD107a), TNF and IFN-γ were assessed by flow cytometry. 16, 20, 26 Data were acquired on a Gallios flow cytometer (Beckman Coulter) and analyzed using FlowJo software (Tree Star v10.6; RRID:SCR_008520).
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