CD8 T cells were purified from the spleens of adult P14 mice via negative selection by antibody depletion of B220 (RA3-6B2, BD Pharmingen), CD11c (HL3, BD Pharmingen), CD11b (M1/70, BD Pharmingen), CD16/32 (2.4G2, BD Pharmingen), IA/IE (2G9, BD Pharmingen) and CD4 (L3T4 RM4-5, BD Pharmingen) with magnetic bead separation (anti-rat IgG Dynal beads, Invitrogen). CD4 T cells were purified from the spleens of adult Smarta mice via negative selection by antibody depletion of B220 (RA3-6B2, BD Pharmingen), CD11b (M1/70, BD Pharmingen), CD16/32 (2.4G2, BD Pharmingen), IA/IE (2G9, BD Pharmingen), CD8α (Clone 53–6.7, BD Pharmingen) with magnetic bead separation (anti-rat IgG Dynal beads, Invitrogen). Cells were counted and labeled with CellTraceTM Violet proliferation dye (Invitrogen) according to manufacturers instructions. FACS sorted DCs were plated at 1×104 cells/well of a round bottom 96 well plate together with 1×105 cells/well of purified CD8 T cells or 3×104 CD4 T cells in RPMI 1640 (Invitrogen) supplemented with 5 mM Hepes, 10% FBS (HyClone), 1% Penicillin Streptomycin (Life Technologies), 1% Glutamax (Life Technologies) and 50μM ≤β-mercaptoethanol (Life Technologies). As positive control for T cell proliferation, T cells were co-cultured with GP33 or GP61 (Abgent) peptide pulsed naïve CD8αneg DCs.
Cd16 32 2.4g2
Manufactured by BD
Sourced in United States
The CD16/32 (2.4G2) is a laboratory instrument used to identify and analyze specific cell surface receptors. It functions as a monoclonal antibody that binds to the Fc gamma receptor III (CD16) and the Fc gamma receptor II (CD32), which are expressed on various immune cells. This product can be utilized in flow cytometry and other immunological applications to study the expression and distribution of these receptors.
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Lab products found in correlation
Lsrfortessa, by BD (4 mentions)
Cd11b m1 70, by BD (2 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Cd8 pe, by BD (2 mentions)
Ovalbumin (ova), by Merck Group (2 mentions)
5 bromo 2 deoxy uridine labeling and detection kit 3, by Roche (2 mentions)
Collagenase d, by Roche (2 mentions)
Fitc or pe conjugated monoclonal antibodies abs, by BD (2 mentions)
Non labeled abs, by BD (2 mentions)
Fixation permeabilization solution, by Thermo Fisher Scientific (2 mentions)
Foxp3 fjk 16s, by Thermo Fisher Scientific (2 mentions)
Ly6g 1a8, by Thermo Fisher Scientific (2 mentions)
Lsr 2 system, by BD (2 mentions)
Cd45 30 f11, by Thermo Fisher Scientific (2 mentions)
Foxp3 staining buffer kit, by Thermo Fisher Scientific (2 mentions)
Facscan, by BD (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Anti rat igg dynal beads, by Thermo Fisher Scientific (1 mentions)
22 protocols using cd16 32 2.4g2
1
CD8/CD4 T Cell Purification and Activation
2
Isolation and Flow Cytometry Analysis of Immune Cells
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Cells from spleen and lymph nodes were isolated by grinding the organ through a 40-µm-pore-size nylon cell strainer to create single-cell suspensions. Splenocytes were treated with red blood cell (RBC) lysis buffer to remove contaminating RBCs. Single-cell suspensions were stained with fluorescently labeled antibodies: CD3 (clone 145-2C11), CD4 (GK1.5), CD8 (5H10-1), CD45R (RA-6B2), NK1.1 (PK136), CD69 (H1.2F3), F4/80 (BM8), Ly6G (1A8), and CD16/32 (2.4G2) (purchased from BD Biosciences or BioLegend). These antibodies were purified or conjugated with peridinin chlorophyll protein (PerCP)/Cy5.5, fluorescein isothiocyanate (FITC), allophycocyanin (APC)/Cy7, APC, phycoerythrin (PE)-Cy7, PE, or BV650. Isotype controls were used as negative controls. Live cells were discriminated with a fixable Live/Dead stain (Life Technologies). Stained cells were analyzed by flow cytometry on a BD LSR Fortessa (BD Biosciences), and data were analyzed with FlowJo software (Tree Star, Inc.).
3
Multiparametric T Cell Phenotyping
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Fc receptors were blocked with purified anti–mouse CD16/32 (2.4G2; BD). The cells were suspended in FACS buffer and stained at saturating conditions with the anti–mouse monoclonal antibodies against CD3 (145-2C11; eBioscience), CD4 (RM4-5; Invitrogen), CD44 (1M7; eBioscience), CD8 (53–6.7; eBioscience), PD-1 (RMP1-30; BioLegend), KLRG1 (2F1; BioLegend), Ly6C (HK1.4; eBioscience), CD62L (MEL-14; eBioscience), CD127 (A7R34; eBioscience), and CD43 (S7; BD). To exclude cells that may have nonspecifically bound to the tetramers, antibodies against non–T cell markers F4/80 (BM8; eBioscience), CD19 (eBio1D3; eBioscience), CD11c (N418; eBioscience), and CD11b (M1/70; eBioscience) were included in a dump channel. All surface staining was done at 4°C for 30 min, except staining for CXCR3 (CXCR3-173; eBioscience), CXCR5 (2G8; BD), and CCR7 (4B12; eBioscience), which was done at room temperature for 1 h. Samples were fixed in PBS containing 2% paraformaldehyde.
4
Leukemic Cell Culture Characterization
5
Flow Cytometry Analysis of Immune Cell Populations
6
Flow Cytometry Analysis of Immune Cell Populations
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Cells present in infected ears and draining lymph nodes were extracted at 7 d post-infection (p.i.) and analysed by flow cytometry as described [49 (link)]. Splenocytes and lymph node suspension cells were obtained by forcing the organ through a stainless steel mesh. Splenocytes were treated with red blood cell (RBC) lysis buffer to remove contaminating RBCs. Single cell suspensions were stained with fluorescence-labelled antibodies: anti-CD3 (clone 145−2 C11), CD4 (GK1.5), CD8 (5H10-1), CD45R (RA-6B2), NK1.1 (PK136), CD69 (H1.2F3), Ly6G (1A8), F4/80 (BM8) and CD16/32 (2.4G2) antibodies were purchased from BD Biosciences or from Biolegend. These antibodies were purified or conjugated with PerCP/cy5.5, FITC, APC/Cy7, APC, PE-Cy7, PE, or BV650. Relevant isotypes were used as control. Flow cytometry was performed with a BD LSR Fortessa (BD Biosciences) and data were analysed with FLOWJO software (Tree Star Inc., Ashland, OR). LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies, Paisley, UK) was used to exclude non-viable cells from analysis.
7
Murine Dendritic Cell Isolation and Stimulation
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Recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (rmIL)-4 were purchased from R&D Systems (Minneapolis, MN, USA), propidium iodide (PI), and ovalbumin (OVA) were purchased from Sigma-Aldrich (Steinheim, Germany), and lipopolysaccharide (LPS) and OVA-Alexa 488 were purchased from Invitrogen (Carlsbad, CA, USA). The following FITC- or PE-conjugated monoclonal antibodies (Abs) and non-labeled Abs were purchased from BD Biosciences (San Jose, CA, USA): FITC-annexin V, CD16/32 (2.4G2), CD11c (HL3), IA[b] (AF6–120.1), IFN-γ, CD4, PE-CD8, CD4. Cytokine ELISA primary and secondary -antibodies specific for murine IL-1β, IL-6, IL-12p70, IFN-γ, IL-2, IL-10, and TNF-α were purchased from BD Biosciences (San Jose, CA, USA). 5-Bromo-2′-Deoxy-Uridine Labeling and Detection Kit III and collagenase D were purchased from Roche (Salt Lake City, UT, USA).
8
Murine Immune Cell Phenotyping
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The above-referenced collected LPLs were washed with PBS twice and gently resuspended in PBS containing 5% FBS for 15 min in the dark at 4 °C with the blocking Fc-receptor CD16/32 (2.4G2, 1:50, BD Biosciences, San Diego, CA, USA). The cells were then incubated with labeled antibodies for 30 min at 4 °C (BD Biosciences, Franklin Lakes, NJ, USA). The samples were then washed twice with PBS containing 5% FBS. The samples were immediately analyzed or fixed in PBS containing 2% paraformaldehyde. The antibodies (Abcam, Inc., Cambridge, UK) used for analysis were: FITC-conjugated anti-mouse Ly6G (1A8), Ly6C (HK1.4), or CD11b (M1/70). The cells were analyzed via flow cytometry (BD Bioscience, San Diego, CA, USA) and processed using FlowJo software (Tree Star, Ashland, OR, USA).
9
Murine Lymphocyte Cytokine Profiling
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OVA and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (Steinheim, Germany). HPLC grade water and acetonitrile were purchased from Daejung Chemical (Siheung-si, Gyeonggi-do, Republic of Korea). Spin-X centrifuge filter 0.45 μm with cellulose acetate was purchased from Costar (Corning Incorporated, Corning, NY, USA). The following FITC- or PE-conjugated monoclonal anti bodies (Abs) and non-labeled Abs were purchased from BD Bioscience (San Joes, CA, USA): FITC- IFN-γ, PE-CD8, and CD16/32 (2.4G2). The cytokine ELISA primary and secondary–antibodies specific for murine IL-2, IFN-γ were purchased from BD Biosciences (San Jose, CA, USA). The 5-Bromo-2 –Deoxy-Uridine Labeling and Detection Kit III, and collagenase D was purchased from Roche (Salt Lake City, UT, USA).
10
Quantification of Angiogenic Cell Populations
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