To generate a single cell suspension, mouse spleens were passed through a 40 μm filter, and RBCs were lysed in a 0.86% NH4Cl solution. Cells were kept in complete RPMI (RPMI 1640, 10% fetal bovine serum, 10% non-essential amino acids, 10% sodium pyruvate, 10% L-glutamine, 10% penicillin and streptomycin, and 1% 2-βME). Cells were washed in FACS buffer (1× PBS, 0.2% BSA and 0.2% 0.5M EDTA), and Fc receptors were blocked with anti-mouse CD16/32 (24G2; BioXCell) in a buffer containing normal mouse and rat IgG (Life Technologies). Abs against CD4, PD-1, Ter119, CD49d, CD11a, B220, CD3ε, CD11b, CD11c, CD44, CD45.1, IFN-γ, TNF-α, CD138, CD80, CD38, GL-7, IgD, IgM, ICOS, SA-APC, SA-BV510, and fixable viability dye were from ThermoFisher or Tonbo Biosciences. Abs against CXCR5, TCR-β, CD19, B220, CD73, and CD44 were from Biolegend, while CXCR5-biotin and CD62L were from BD Biosciences. Following surface staining, samples not requiring intracellular staining were fixed in 4% PFA (Electron Microscopy Sciences). Fluorescence minus one (FMO) controls were used for setting the positive gates and indicating background staining. Samples were run on an LSRIIFortessa (Becton Dickson), and FlowJo 10.3 was used for analysis.
Anti mouse cd16 32 24g2
Manufactured by BioXCell
Sourced in Germany
Anti-mouse CD16/32 (24G2) is a monoclonal antibody that binds to the mouse CD16 and CD32 receptors. CD16 and CD32 are Fc gamma receptors expressed on various immune cells, including macrophages, dendritic cells, and natural killer cells. This antibody can be used to block the binding of Fc-containing immune complexes to these receptors.
Automatically generated - may contain errors
Lab products found in correlation
Lsrii fortessa, by BD (3 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Celltrace violet ctv cell proliferation kit, by Thermo Fisher Scientific (2 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Annexin 5 binding buffer, by BioLegend (2 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (2 mentions)
Annexin 5 fitc, by BD (2 mentions)
Normal mouse and rat igg, by Thermo Fisher Scientific (2 mentions)
Macs buffer, by Miltenyi Biotec (2 mentions)
Anti pe microbeads, by Miltenyi Biotec (2 mentions)
Automacs pro cell separator, by Miltenyi Biotec (2 mentions)
Sa apc, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Zombie red viability dye, by BioLegend (1 mentions)
Lsr fortessa x 20 sorp, by BD (1 mentions)
Anti gata3 16e10a23, by BioLegend (1 mentions)
Anti mouse human cd11b m1 70, by BioLegend (1 mentions)
Anti mouse f4 80 bm8, by BioLegend (1 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
6 protocols using anti mouse cd16 32 24g2
1
Spleen Cell Isolation and Staining
2
Single-cell Skin Immune Profiling
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Single cell suspensions of ear skin were prepared by incubating dermal side down in 500 uL 0.25 mg/mL Liberase TL for 90 min at 37 °C. After filtration through a 100 µm cell strainer, cell suspensions were stained with ZombieRed viability dye (423109, Biolegend, San Diego, CA, USA) per manufacturer’s instructions and then with conjugated antibodies on ice for 30 min. Stained cells were then washed and fixed with Cytoperm/Cytofix solution (BD Biosciences, San Jose, CA, USA) and acquired on an LSR Fortessa X-20 SORP. Data were analyzed using FlowJo software. anti-mouse CD16/32 (2.4G2) was from BioXCell (West Lebanon, NH, USA) (CUS-HB-197) and used at 1:300 dilution. anti-T-bet (4B10) (644803), anti-GATA3 (16E10A23) (653813), anti-mouse CD4 (GK1.5) (100449), anti-mouse CD3ε (145-2C11) (100327), anti-mouse CD45 (30-F11) (103105, 103111), anti-mouse NKp46 (29A1.4) (137617), anti-mouse/human CD11b (M1/70) (101245), anti-mouse CD11c (N418) (117328), anti-mouse Ly-6G (1A8) (127639), anti-mouse F4/80 (BM8) (123146), anti-mouse I-Ab (AF6-120.1) (116419) were from Biolegend and used at 1:300 dilution.
3
Multi-parameter Flow Cytometry Analysis
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Antibodies and dilutions are described in the reporting summary. For staining, single cell preparations were made from spleen and lymph node in MACS buffer (PBS with 2% FBS and 2 μM EDTA). Staining panels containing anti-CD93 (Supplementary Fig. 1d ) were performed in MACS buffer not supplemented with EDTA. After ACK (Ammonium Chloride) lysis of RBCs, cells were washed once and Fc receptors blocked with anti-mouse CD16/32 (2.4G2, BioXCell). Cells were incubated with antibodies for 45/60 min on ice. Intracellular staining of BCL-6, Foxp3 and MCL-1 were performed using the Foxp3-staining buffer set from eBioscience. The following reagents were used according to the manufacture instructions: PhiPhiLux-G1D2 kit (OncoImmunin Inc.), Annexin V-FITC (BD Biosciences) and Annexin V binding buffer (BioLegend), CellTrace™ Violet (CTV) Cell Proliferation Kit from Life Technologies, LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit and LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit (Life Technologies). The following gates were applied before the identification of the specific cell types: FSC-A/SSC-A, exclusion of doublets (SSC-H/SSC-W and FSC-H/FSC-W), live cells (negative for Aqua or Near-IR dead stain kit); gating strategies for each population are indicated in each figure legend. Flow cytometry was performed on a LSRII (BD Biosciences) and data analyzed using FlowJo 9.9 software (TreeStar).
4
Multi-parameter Flow Cytometry Analysis
5
Enrichment of Antigen-Specific T Cells
6
Isolation and Detection of Antigen-Specific T Cells
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Single-cell suspensions of splenocytes were prepared as described above. Following red blood cell lysis, cells were resuspended in MACs buffer (Miltenyi BioTec, Bergisch Gladbach, Germany), and Fc receptors were blocked with anti-mouse CD16/32 (24G2; BioXCell) in a buffer containing normal mouse and rat IgG (ThermoFisher) for 20 minutes at 4 o C. Then 10 nM of the prepared decoy reagent was incubated with the cells for 5 minutes at room temperature.
MSP-1 or AMA-1-PE tetramers were then added at a concentration of 10 nM of SA, and the cells were incubated at 4 o C for 30 minutes. Cells were subsequently washed in MACs buffer before incubating with anti-PE microbeads (Miltenyi) for 5 minutes at 4 o C. Finally, cells were washed again in MACs buffer prior to positive selection on an AutoMACS Pro cell separator (Miltenyi). Recovered cells were surface stained and ran on a BD LSRIIFortessa. Collected data were analyzed using FlowJo version X software.
MSP-1 or AMA-1-PE tetramers were then added at a concentration of 10 nM of SA, and the cells were incubated at 4 o C for 30 minutes. Cells were subsequently washed in MACs buffer before incubating with anti-PE microbeads (Miltenyi) for 5 minutes at 4 o C. Finally, cells were washed again in MACs buffer prior to positive selection on an AutoMACS Pro cell separator (Miltenyi). Recovered cells were surface stained and ran on a BD LSRIIFortessa. Collected data were analyzed using FlowJo version X software.
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