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2 protocols using cd11c alexa fluor 700 clone hl3

1

Immunophenotyping of Colonic Leukocytes

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Large intestine samples were removed, open longitudinally, and gently washed with ice-cold PBS. Tissue was cut into small pieces and washed twice in predigestion solution (1X HBSS, 5 mM EDTA, 1 mM DTT) for 20 min at 37 °C with gentle shaking. Tissue was then enzymatically digested in collagenase D (0.05 g/100 ml PBS). Collected fractions were pooled within sample and lysed to remove red blood cells. Staining of single-cell suspensions included the following antibodies from BD: Ly6C (FITC; clone AL-21), CD11b (PerCP-Cy5.5; clone M1/70), Siglec F (BV421; clone E50-2440), CD11c (Alexa Fluor 700; clone HL3), CD45.2 (BUV395; clone 30-F11), EpCAM (PE; clone G8.8), Ly6G (PE-Cy7; clone 1A8), and MHCII (BV711; clone M5/11.415.2). CD169 (eFluor 660; clone SER-4) was purchased from eBioscience, and F4/80 (APC/Cy7; clone BM8) was purchased from BioLegend. DAPI (Life Technologies) was used as a viability control. Samples were collected on BD LSRFortessa X-20 with the use of BD FACSDiva v.8.0 software for data acquisition, analyzed using FlowJo v.10.2 software, and enumerated by back calculating based off of percentages relative to total cell counts.
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2

Comprehensive Immunological Protocol for Cell Analysis

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Resveratrol (⩾99% pure), olive oil (highly refined, low acidity) and DMSO were purchased from Sigma-Aldrich (St Louis, MO, USA). F(ab')2 fragment of goat anti-mouse IgG, μ-chain-specific goat anti-mouse IgM, and rabbit peroxidase-anti-peroxidase ICs were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Mouse mAb specific for CD19-PE-Cy7 (clone 1D3) was obtained from eBioscience (San Diego, CA, USA). Mouse IgG isotypes and mAbs specific to CD16/32 (clone 2.4G2), CD138-BV421 (clone 281-2), CD11b-PerCP-Cy5.5 (clone M1/70), CD11c-AlexaFluor 700 (clone HL3) and GL7-AlexaFluor 647 (clone GL7) were acquired from BD Biosciences (San Jose, CA, USA). Mouse IgM isotype Ab was obtained from SouthernBiotech (Birmingham, AL, USA). Ninety-six-well MultiScreen HTS filter plates were acquired from Merck Millipore (Billerica, MA, USA). Blood lancet was obtained from MEDIpoint (Mineola, NY, USA). Vectastain ABC kits packed with biotinylated goat anti-rabbit IgG, rabbit anti-goat IgG and rabbit anti-rat IgG mAbs were purchased from Vector Laboratories (Burlingame, CA, USA). Abs specific to mouse B220, FcγRIIB, and phosphor-c-Abl (Y245) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Urine analysis strips were acquired from Macherey-Nagel (Duren, Germany).
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