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

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.

1. Draw 200 mL of blood under regulated conditions into a tube containing heparin.
2. Add 15 mL of the density gradient medium into a 50 mL tube with a porous barrier incorporated. Spin the tube at 1000 x g for 30 s.
3. Add 12....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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