Sep 01, 2022

Public workspaceProliferation assay

DOI
dx.doi.org/10.17504/protocols.io.261geojwol47/v1
  • 1University of Minnesota
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DOI: dx.doi.org/10.17504/protocols.io.261geojwol47/v1
External link: https://doi.org/10.3389/fimmu.2023.1060905
Protocol Citation: Philippa R Kennedy 2022. Proliferation assay. protocols.io https://dx.doi.org/10.17504/protocols.io.261geojwol47/v1
Manuscript citation:
Kennedy PR, Vallera DA, Ettestad B, Hallstrom C, Kodal B, Todhunter DA, Bendzick L, Hinderlie P, Walker JT, Pulkrabek B, Pastan I, Kratzke RA, Fujioka N, Miller JS, Felices M, A tri-specific killer engager against mesothelin targets NK cells towards lung cancer. Frontiers in Immunology doi: 10.3389/fimmu.2023.1060905
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 20, 2020
Last Modified: September 01, 2022
Protocol Integer ID: 35954
Abstract
Assessing the impact of drugs and treatments on natural killer (NK) cell viability and expansion.
PBMCs or enriched NK cells (see Isolating NK cells from human blood products) are labeled with a permanent amine-reactive dye (CellTrace Violet Proliferation Kit, Cat. No:C34557, Thermo Fisher) according to the manufacturer's instructions.
Cells are exposed to various experimental conditions, then harvested after 7 days of culture.
Optional: If required, cells can be stained for apoptotic markers (FITC Annexin V, Cat. No: 556419, BD Biosciences) according to the manufacturer's instructions prior to antibody staining.
Cells are stained with a dead cell marker (LIVE/DEAD NEAR-IR, Cat. No: L34976, Thermo Fisher), PE-CY7 conjugated anti-CD56 (HCD56, BioLegend), and PE-CF594 conjugated anti-CD3 (UCHT1, BD BioSciences).
The samples are run on a flow cytometer (LSRII, BD Biosciences) and analyzed using FlowJo software (Tree Star Inc., RRID:SCR_008520).

The proliferation of live (dead cell marker- and annexin V-) NK cells (CD56+/CD3-) and T cells (CD56-/CD3+) is assessed by dilution of the dye.

Relative numbers of live NK cells and T cells for each treatment condition are obtained by running the flow cytometer at a constant flow rate and analyzing the number of each cell type obtained within a specified time limit e.g. 60s.