An In Vitro Artificial Activation Assay for Studying Fc Receptor Activation by Antibodies

Published: March 29, 2024

Abstract

Source: Petriello, A. et al. Assessment of Human Natural Killer Cell Events Driven by FcγRIIIa Engagement in the Presence of Therapeutic Antibodies. J. Vis. Exp. (2020)

This video demonstrates FcγRIIIa-driven events initiated by therapeutic antibodies in human natural killer cells. The artificial stimulation platform facilitates the exploration of downstream effector functions, encompassing cytoskeletal rearrangement, degranulation, chemokine/cytokine production, and signaling pathways mediated by the FcγRIIIa and Fc portions of antibodies involved in binding.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Isolation of PBMCs and enrichment/purification of NK cells NOTE: Other methods for the isolation of peripheral blood mononuclear cells (PBMCs) and enrichment/purification can also be performed. Draw 200 mL of blood under regulated conditions into a tube containing heparin. Ad…

Divulgaciones

The authors have nothing to disclose.

Materials

16% paraformaldehyde Thermo Fisher Scientific 50-980-487
Alexa Fluor 488 phalloidin Thermo Fisher Scientific A12379
anti-mouse HRP antibody Cell Signaling Technologies 7076
AutoMACS instrument Miltenyi NK cell isolation method; another isolation instrument may be used
CD107a APC antibody Biolegend 328620
Bovine Serum Albumin Fraction V, fatty acid free Millipore Sigma 10775835001
Cytokine 30-Plex Human Panel Thermo Fisher Scientific LHC6003M chemokine/cytokine method; another chemokine/cytokine analysis method may be used
FBS (Fetal Bovine Serum) Hyclone SH30071.01
Goat anti-human κ light chain
antibody
Millipore Sigma AP502
Halt Protease and Phosphatase Inhibitor Cocktail Thermo Fisher Scientific 78440
pAKT (S473) antibody Cell Signaling Technologies
pERK1/2 antibody Cell Signaling Technologies
pPRAS40 antibody Cell Signaling Technologies 13175
β-actin HRP antibody Abcam ab6721
NK cell isolation kit Miltenyi 130-092-657 NK cell isolation kit; another isolation kit may be used

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An In Vitro Artificial Activation Assay for Studying Fc Receptor Activation by Antibodies. J. Vis. Exp. (Pending Publication), e22089, doi: (2024).

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