Spleens were isolated from euthanized naïve BLT donor mice and mechanically disrupted in sterile PBS 2% FBS using frosted glass slides to generate a single cell suspension. Immune cells were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare) as per the manufacturers protocol (950 g, 20 min, no brake). Immune cells were washed once in sterile PBS supplemented with 2% FBS, re-suspended in 150 microliters (ul) PBS 2% FBS, incubated with murine and human Fc-Block (BD) for 10 minutes at 4°C, and stained with antibodies specific to murine CD45 (30-F11; Biolegend) and human CD43 (CD43–10G7; Biolegend). CD43- target cells (B cells and dendritic cells (DC)) were isolated using Fluorescence Activated Cell Sorting (FACS) on a BD ARIA, resulting in ≥ 99.8% purity.
Murine and human fc block
Manufactured by BD
Sourced in United States
Murine and human Fc-Block is a laboratory reagent that blocks Fc receptors on cells, preventing non-specific binding of antibodies. It is used to reduce background signal and improve specificity in immunoassays and other applications involving antibody detection.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using murine and human fc block
1
Isolation of Murine Immune Cells
2
Isolation of Murine and Human NK Cells
3
Humanized NSG-Tg(IL-15) Mouse Model
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NOD.Cg-PrkdcscidIl2rgtm1wjl Tg(IL-15) mice (NSG-Tg(IL-15) (Stock No: 030890)) were developed in Dr. Leonard Shultz’s research lab by pronuclear injection of a ~200 Kbp bacterial artificial chromosome (BAC) designated Chori BACPAC RP11-620F3 containing the human IL-15 gene. NSG-Tg(IL-15) mice (Stock No: 030890), CD34+ humanized NSG-Tg(IL-15), and NSG mice were provided by or purchased from The Jackson Laboratory. Recipient mice were sublethally irradiated at 4–12 weeks of age and intravenously injected with CD34+ enriched human cord-blood-derived cells. The reconstitution efficiency of each animal was independently determined by flow cytometric analysis of PBMCs from whole blood collected from the submandibular vein. PBMCs were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Chicago, IL, USA) as per the manufacturer’s protocol. Cells were washed and resuspended in 0.2 mL of PBS supplemented with 2% FBS, incubated with murine and human Fc Block (Becton Dickinson (BD), Franklin Lakes, NJ, USA) for 10 min at room temperature (RT), and then stained with antibodies specific to murine CD45 (30-F11; BioLegend, San Diego, CA, USA) and human CD45 (H130; BioLegend, San Diego, CA, USA), before acquisition through flow cytometry.
4
Multiparametric Analysis of Immune Cells
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Blood, spleen, liver, bone marrow (BM), and the gastrointestinal (GI) tract were harvested at experimental endpoints. Single-cell suspensions were generated by mechanical disruption using a 40 μm nylon mesh. Immune cells were then isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Chicago, IL, USA) as per the manufacturer’s protocol. Immune cells were washed once in sterile PBS + 2% FBS, resuspended in PBS + 2% FBS, incubated with murine and human Fc Block (BD) for 10 min at RT, and stained with antibodies listed in Supplementary Table S1 . For permeabilization, the FoxP3 permeabilization buffer kit (Tonbo Biosciences, San Diego, CA, USA) was used according to the manufacturer’s instructions. The acquisition was performed on the 5 L 16UV-16V-14B-10YG-8R Cytek™ Aurora flow cytometer (Cytek Biosciences, Fremont, CA, USA). Analyses of FCS files were performed using FlowJo (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Data were graphed and statistical analyses were performed using Graphpad Prism (San Diego, CA, USA).
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