After infection, primary monocytes and THP‐1 cells were washed with phosphate buffer solution (PBS) and fixed with 3% paraformaldehyde at 4°C. Blocking of FcγRIIA was performed with goat polyclonal CD32A (1:100, R&D Systems AF1875) for 30 min at 4°C. After washing with PBS, mouse anti‐CD32 IV.3 (1:300, Stem Cell Technologies 60012) was then added for 30 min at 4°C. After further washing with PBS, anti‐mouse AlexaFluor 488 (1:400) was added and incubated at 4°C for 30 min prior to analysis with the BD LSRFortessa flow cytometer. In a separate experiment, primary monocytes and THP‐1 cells cultured at 20% O2 and 3% O2 for 24 h were washed with PBS and fixed with 3% PFA. Mouse anti‐FcγRI (1:300, Abcam ab46679) and mouse anti‐CD32 IV.3 (1:300, Stem Cell Technologies 60012) antibodies were added for 30 min at 4°C. After washing with PBS, anti‐mouse AlexaFluor 488 (1:400) was added and incubated at 4°C for 30 min prior to analysis with the BD LSRFortessa flow cytometer.
Af1875
Manufactured by R&D Systems
AF1875 is a recombinant human protein. It is a member of the TGF-beta superfamily of proteins.
Automatically generated - may contain errors
2 protocols using af1875
1
Monocyte FcγR Expression Analysis
2
Investigating Interferon-α Modulation of CMP-001 Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
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PBMCs were isolated, diluted to 1×106 cells/mL and treated with 10 μg/mL anti-Qβ-coated CMP-001 with or without antihuman IFN-α (5 μg/mL, MMHA-2, Cat #211002; Thermo Fisher), antihuman IFNAR2 (5 μg/mL, MMHAR-2; PBL; Cat #21 385–1), mouse IgG2a, kappa isotype control (5 μg/mL, Cat# 16-4724-85; Thermo Fisher), or mouse IgG1, kappa isotype control (5 μg/mL, Cat #14471482; Thermo Fisher) for 24 hours. For CD32a neutralization experiments, monocytes were diluted to 1×106 cells/mL and treated with 50 μg/mL anti-CD32a (R&D, #AF1875) or isotype control (R&D, #AB-108-C) for 15 min followed by treatment with 10 μg/mL anti-Qβ-coated CMP-001 and 1.792×104 U/mL recombinant IFN-α for 18–20 hours.
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