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…
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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 |
