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Cd32 pe

Manufactured by BD
Sourced in United States

The CD32-PE is a laboratory instrument used for the detection and analysis of specific cellular markers. It employs flow cytometry technology to identify and quantify the expression of the CD32 protein on the surface of cells. The CD32-PE provides researchers with a precise and reliable tool for immunophenotyping and cellular characterization studies.

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3 protocols using cd32 pe

1

Phenotyping Monocyte Subsets via Flow Cytometry

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Frozen PBMCs were thawed and distributed into three tubes for antibody staining. All tubes contained antibodies to the following markers: CD14-FITC (Southern Biotechnology Associates), CD16-AF700 (BD Bioscience), and CD3- and CD19-PerCP.Cy5.5 (eBioscience). Additional antibodies included: Tube 1, CD11b-APC-Cy7 (eBioscience) and CD32-PE (BD Bioscience); Tube 2, CD64-PE (BD Bioscience); and Tube 3, CD32B-PE detected with goat anti-rabbit PE (AbCAM). Proper isotype controls were included for each primary antibody. All incubations were done in ice. Fc-receptors were blocked with human IgG (Sigma-Aldrich) for 20 min, followed by addition of antibodies for 30 min in PBS with 1% bovine serum albumin, 0.1 mM EDTA and 0.02% sodium azide. Prior to analysis 7AAD (BD Bioscience) was added to each tube. Acquisition was conducted in a FACS CANTO-II using FACS DIVA v6.0 (BD Biosciences). Monocytes were identified based on their forward and side scatter properties and then dead (7AAD-positive), CD3 and CD19-positive cells were excluded. Further details on the gating strategy are provided in the Supplement (Fig. S2). After identifying monocyte sub-populations based on CD14 and CD16 expression, the median fluorescence intensities of each fluorochrome was established.
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2

Flow Cytometric Analysis of Cell Markers

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Flow cytometric analysis was performed on Guava-easyCyte (6HT) and BD FACSCalibur flow cytometry systems, using InCyte or FlowJo software for data acquisition and analysis. The antibodies for staining human cell surface markers VE-cadherin-FITC, CD43-PE, CD45-APC and CD32-PE were obtained from BD Biosciences, NJ, USA.
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3

Microglia Phenotyping by Flow Cytometry

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Isolated microglia were suspended in an incubation buffer (50 μl; 1 × PBS + 0.1%BSA) for 30 min on ice then Fc receptors blocked with anti-CD32 (BD Bioscience, San Jose, CA). Fluorescent conjugated antibodies were applied on ice for 30 min in the dark to assess microglia purity (mouse anti-rat CD11b-FITC, BD Pharmingen, San Jose, CA; mouse anti-rat-CD45-APC, eBioscience, San Diego, CA) and state of activation (mouse anti-rat: MHC-II-PE, CD32-PE, CD86-PE; BD Bioscience, San Jose, CA). For CD206, cells were incubated in rabbit anti-rat CD206 then donkey anti-rabbit-PE secondary antibody (BD Bioscience, San Jose, CA). Following washes in PBS, cells were analyzed on an Attune Acoustic Focusing Cytometer (ABI, Carlsbad, CA) calibrated with commercially available beads prior to each run. Fluorescence spillover compensation values were generated from non-stained cell populations and single-color staining controls. Isotype controls were used to exclude the non-specific binding of antibodies. For each staining condition, 1 × 104 events were collected.
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