CD14+ monocytes were cocultured with MSCs using Polycarbonate Membrane Transwell® Inserts (0.4-µm pores). CD14+ monocytes (2 × 105) were seeded in the lower chambers, and MSCs (2 × 104) were seeded in the upper chambers. Recombinant human M-CSF (25 ng/ml, PeproTech, New Jersey, USA) was added to activate monocytes. Five days later, lipopolysaccharide (LPS; 50 ng/ml, SigmaAldrich, Darmstadt, Germany) and recombinant human interferon-γ (IFN-γ; 20 ng/ml, PeproTech) were added to induce macrophage polarization to the M1 phenotype, and recombinant human IL-4 (20 ng/ml, PeproTech) and IL-10 (20 ng/ml, PeproTech) were added to induce macrophage polarization to the M2 phenotype. Twenty-four hours later, the phenotype of the macrophages was determined by flow cytometry. Macrophages were digested, incubated with anti-HLA-DR-PE antibody or anti-CD206-BV421 antibody (BD Biosciences, California, USA) and then incubated with fixation medium (Invitrogen, Massachusetts, USA) for 15 min. After three washes with PBS, the cells were incubated with permeabilization medium plus an anti-CD68-FITC antibody (BD Biosciences) for 30 min.
Anti hla dr pe antibody
Manufactured by BD
Sourced in United States
The Anti-HLA-DR-PE antibody is a laboratory reagent used for the identification and analysis of cells expressing the HLA-DR antigen. It is conjugated with the fluorescent dye Phycoerythrin (PE) for detection purposes.
Automatically generated - may contain errors
2 protocols using anti hla dr pe antibody
1
Monocyte-Macrophage Polarization Assay
2
CRISPR-Cas9 Knockout of CIITA Exon 3
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CRISPR/Cas9 guide sequences were identified and double-stranded guide oligos cloned into the pX330 wild-type Cas9 vector as previously described (51 (link)). Guide sequences were chosen to induce a 50 bp deletion spanning CIITA exon 3. Five nanograms each of pX330.CIITAex3.1 and pX330.CIITA.ex3.2 were nucleofected into Raji cells. 7-days post-nucleofection, limiting dilution single-cell cloning of the CRISPR/Cas9 CIITA pool was performed in 96-well plates using 3 dilutions of 3, 1 and 0.3 cells/well. Fourteen days later, wells with positive growth were stained with anti-HLA-DR-PE antibody (BD Biosciences) and screened for loss of HLA-DR surface expression by flow cytometry on an LSRII instrument. One single-cell clone was MHC-II negative (CIITAΔex3). Deletion of CIITA exon 3 was confirmed by PCR amplification across the deleted exon followed by TOPO TA cloning (Life Technologies) and sequencing of three independent colonies. All colonies contained the exact same 50 bp deletion at a position denoted by the PAM sequence. CRISPR/Cas9 guide oligos used for cloning and PCR primers spanning CIITA exon 3 are listed in Supplemental Table S2.
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