Primary antibodies used for IHC and flow cytometry were as follows: c-Kit (2B8; eBioscience), CD16/32 (93; eBioscience), VE-cadherin (eBioscience), c-Kit (R&D Systems), Sca-1 (E13-161.7; BioLegend), CD48 (HM48-1; BioLegend), CD150 (TC15-12F12.2; BioLegend), IL-7Rα (SB/199; BioLegend), endoglin (MJ7/18; BioLegend), CD4 (L3T4; BD), CD8 (53-6.72; BD), B220 (RA3-6B2; BD), TER-119 (BD), Gr-1 (RB6-8C5; BD), CD34 (RAM34; BD), Mac-1 (M/70; BD), Flt-3 (A2F10.1; BD), CD41 (MWReg30; BD), CD45.2 (104; BD), CD45.1 (A20; BD), GPIbα (Xia.G5; Emfret), Clec2 (AbD Serotec), and Thpo (Bioss). Secondary antibodies for IHC were Alexa Fluor 488–conjugated IgGs (Molecular Probes) or Cy3/Cy5/DyLight549/DyLight649-conjugated IgGs (Jackson ImmunoResearch Laboratories, Inc.). IHC specimens were treated with DAPI (Molecular Probes) for nuclear staining. Stimulatory rabbit anti–mouse CLEC-2 antibody was a gift of K. Suzuki-Inoue.
Cd16 32
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CD16/32 is a laboratory equipment product designed for cell surface marker analysis. It functions as a Fc-receptor blocker, which is used to prevent non-specific binding during flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b, by Thermo Fisher Scientific (6 mentions)
Sca 1, by Thermo Fisher Scientific (4 mentions)
H 2kd, by Thermo Fisher Scientific (3 mentions)
H 2kb, by Thermo Fisher Scientific (3 mentions)
Anti mouse cd3, by Thermo Fisher Scientific (3 mentions)
Ter119, by Thermo Fisher Scientific (3 mentions)
C kit 2b8, by Thermo Fisher Scientific (2 mentions)
Sca 1 e13 161.7, by BioLegend (2 mentions)
Cd48 hm48 1, by BioLegend (2 mentions)
Cd4 l3t4, by BD (2 mentions)
Cd41 mwreg30, by BD (2 mentions)
Cd45.1 a20, by BD (2 mentions)
B220 ra3 6b2, by BD (2 mentions)
F4 80, by BD (2 mentions)
Pan t isolation kit, by Miltenyi Biotec (2 mentions)
Regulatory t cell isolation kit, by Miltenyi Biotec (2 mentions)
Anti cd90.2 microbeads, by Miltenyi Biotec (2 mentions)
Foxp3 pe, by BioLegend (2 mentions)
Cd25 apc, by BioLegend (2 mentions)
Cd150, by Thermo Fisher Scientific (2 mentions)
48 protocols using cd16 32
1
Immunophenotyping of Hematopoietic Cells
2
Multiparameter Flow Cytometry of Immune Cells
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Spleen and lymph nodes were isolated, and single-cell suspensions were obtained by crushing the tissues through 70 µm filters. The spleen and blood samples were subjected to red blood cell lysis (red blood cell lysis buffer: 8.4 g of NH4Cl and 0.84 g of NaHCO3 per liter of distilled water). Single cell suspensions were stained with fluorescently labeled antibodies (CD45, CD3, CD4, CD8, F4/80, Ly6C, Ly6G, MHCI, MHCII, CD44, CD62L, CD19, CD11C, NK1.1, all from BD Biosciences, San Diego, CA, USA). Nonspecific binding was prevented by the pre-incubation of the cells with a Fc receptor-blocking antibody CD16/32 (Ebioscience, Vienna, Austria). Flow cytometry was performed on a BD Canto II (BD Biosciences), and analysis was performed using the Flowjo software version 10 (Tree star, Ashland, OR, USA).
3
Tumor-Infiltrating Lymphocyte Isolation
4
Murine Hematopoietic Cell Analysis
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TRI reagent was from Sigma-Aldrich (Madrid, Spain). Antibodies for the detection of murine CD135 (Flt3), CD34, CD16/32, and CD127 (IL-7Rα) were from eBioscience (Barcelona, Spain). The Lineage Cell Depletion Kit for mice, FcR Blocking reagent and murine flow cytometry antibodies (CD3e, CD4, CD8a, CD11b, CD19, CD43, CD45R (B220), CD117 (c-Kit), anti-Gr1, anti-Sca-1, anti-Ter-119, anti-IgM) were from Miltenyi Biotec (Madrid, Spain). Super Script II reverse transcriptase and RNase OUT ribonuclease inhibitor were from Invitrogen and Thermo Fisher Scientific (Madrid, Spain). GoTaqR qPCR master mix was from Promega (Madrid, Spain).
5
Regulatory T Cell Isolation Protocol
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Pan T isolation kits, regulatory T cell isolation kits, and anti-CD90.2 microbeads were purchased from Miltenyi Biotec. Antibodies including anti-mouse CD3, TCRβ, CD4, CD8, CD25, H-2Kb, H-2Kd, and CD16/32 were purchased from eBioscience.
6
In Vivo Uptake of Tumor Cells
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C57BL/6 female mice (6–9 weeks old) were injected intra-peritoneum with PBS containing 3 × 106 B16-F10 cells (BNIP3WT or BNIP3KD) pHrodo-labelled (Life Technologies, P36600). The peritoneal cells were collected 24 h post-injection via peritoneal lavage with Ca2+- and Mg2+-free PBS. The cells were transferred to an ultra-low attachment V-bottom plate and stained with the fixable Live/Dead Yellow stain (Invitrogen). After blocking the Fc receptor (CD16/32, eBioscience, 16-0161-82), cells were stained for F4/80-eFluor780 (eBioscience, 47-4801-80), CD11b-eFluor450 (eBioscience, 48-0112-82), MHCII-FITC (eBioscience, 11-5321-81), CD86-APC (eBioscience, 17-0862-81), CD206-PeCy7 (eBioscience, 25-2061-80) in FC buffer. Samples were acquired on a Gallios™ flow cytometer and the data analysis was performed via FlowJo_V10™ software.
7
Phenotypic Analysis of Murine Regulatory T Cells
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Half a million splenocytes were stained in each FACS tube. First, cells were washed with staining buffer (Biolegend, San Diego, CA, USA), and centrifuged at 800 g for 5 min at 4 °C. They were then stained with fluorochrome-conjugated antibodies after blocking of Fc receptors (CD16/32, Biolegend, San Diego, CA, USA) for 5 minutes. The following antibodies were used: FoxP3-PE, CD3-PE/Cy7, CD25-APC, CD4-PB, CD8a-FITC and CD3-PE-Cy7, all from Biolegend (San Diego, CA, USA). The cells were stained 30 min on ice in the dark with antibodies binding extracellular targets. Cells were then washed to remove unbound antibodies, incubated with Fixation/Permeabilization buffers (eBioscience, San Diego, CA, USA) for 30 min on ice and blocked with CD16/32 prior incubation with anti-FoxP3 for 30 min. Cells were resuspended in PBS buffer containing 1% BSA and 0.5 mM EDTA. Cells were run on Gallios flow cytometer (Beckman Coulter, High Wycombe, UK) and analyzed with FlowJo software V.10 (Tree Star, Inc. Ashland, OR, USA).
8
Murine Hematopoietic Stem Cell Isolation
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Bone marrow cells were harvested by crushing two tibias, two femurs, two pelvises, and one spine from each mouse. Bone marrow cells were enriched for immature cells using anti–mouse CD117 MicroBeads and an autoMACS machine (both Miltenyi Biotec) per manufacturer’s instructions. c-Kit–enriched populations were stained with antibodies against lineage markers (B220, CD3, Gr-1, Mac-1, and Ter119), c-Kit, Sca-1, CD150, CD16/32, and CD34 (eBioscience) as previously described (McGowan et al., 2011 (link)). Stained samples were either analyzed or sorted using a FACSAria II (BD). For all experiments in which c-Kithi or c-Kitlo HSCs were purified, they were double-sorted to ensure >95% purity. To calculate the frequency of hematopoietic precursors, two femurs and two tibias were flushed into 1x PBS containing 2.5% fetal calf serum (Hyclone). Bone marrow aspirations were performed on mice under isoflurane anesthesia per IACUC-approved protocol. Aspirates were treated with ACK lysis buffer and stained in PBS/2.5% fetal calf serum with antibodies against lineage markers, c-Kit, Sca-1, CD150, CD16/32, Flk2, CD34, CD48, and CD41 for analysis on hematopoietic stem and progenitor cells. For myeloid progenitors, bone marrow cells were stained with antibodies against lineage markers, c-Kit, Sca-1, CD150, CD16/32, CD41, CD105, and CD71, as described by Pronk et al. (2007) (link).
9
Regulatory T Cell Isolation Protocol
10
Murine Hematopoietic Cell Immunophenotyping
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Cells were isolated from bone marrow, spleen, and thymus of mice, and stained with the following antibodies: B220 (APC; BD Pharmingen), cKit (PE-Cy7; BioLegend, San Diego, CA), CD3 (PE-Cy7; BD Bioscience), CD4 (APC-H7; BD Pharmingen), CD8 (ECD; Beckman Coulter), CD11b (PE; BD Bioscience), CD16/32 (PE; eBioscience, San Diego, CA), CD34 (FITC; BD Pharmingen), CD45.2 (PerCP-Cy5.5; BD Pharmingen), CD48 (APC-Cy7; BD Pharmingen), CD127 (ECD; BD Pharmingen), CD150 (PerCP-Cy5.5; BioLegend), Gr1 (APC-Alexa700; BD Bioscience), Lineage Cocktail (APC; BD Pharmingen), Sca1 (Brilliant Violet 421; BioLegend). Dead cells were excluded using either DAPI or Vivid-Aqua (Invitrogen) staining. All data acquisition was performed on a LSRII (BD) flow cytometer, and results were analyzed using FlowJo v.8.8.7 (TreeStar).
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