Determining the Role of Neutrophil-Like Cells in Lymphoma Cell Sensitivity to a Test Drug

Abstract

Source: Hirz, T., et al. Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents. J. Vis. Exp. (2016).

This video demonstrates an assay investigating the role of neutrophil-like cells in safeguarding lymphoma cells against the cytotoxicity of chemotherapeutic agents. A 3D co-culture, consisting of lymphoma and neutrophil-like cells, is established and exposed to vincristine—a chemotherapy drug. Direct contact with the neutrophil-like cells induces an upregulation of anti-apoptotic proteins in the lymphoma cells, thereby promoting their survival in the presence of vincristine.

Protocol

1. Differentiation of HL60 Cells along the Granulocytic Pathway and Their Coculture with RL Lymphoma B Cells in 3D Model

1. Differentiation of Human Promyelocytic (HL60) Cells into Neutrophil-like Cells
   1. Adjust HL60 cell number to 3 x 10⁵ cells/ml then add 1 µM retinoic acid (RA) and 1.25% DMSO to induce their differentiation. Seed each 3 x 10⁵ HL60 cells per well in 48-well plate then incubate for 48 hr at 37 °C with 5% CO₂.
   2. Later, collect the cells and centrifuge at 300 g for 5 min at RT. Resuspend the cells with complete RPMI medium for counting using cell vi....

Results

Figure 1. Structural Parameters of Differentiated HL60 Cells. (A) Forward-scatter (FSC) and side-scatter (SSC) plots represent HL60 and differentiated HL60 cells (HL60diff). (B) HL60 and HL60diff cells were labe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI 1640	Gibco Invitrogen	21875-034
Fetal bovine serum (FBS)	Gibco Invitrogen	10270-106
Phosphate-buffered saline (PBS contains calcium and magnesium)	Gibco Invitrogen	14040-091
Vincristine	EG labo
BD Matrigel basement membrane matrix	BD Biosciences	354234	Put at 4 oC overnight before the day of the experiment
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D8418
Retinoic acid	Sigma-Aldrich	R2625
Bovine serum albumin (BSA)	Sigma-Aldrich	A7906
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich	E5134
Annexing V-FLOUS staining kit	Roche	11 988 549 001
Kit RAL 555 Modified Giemsa staining kit	Cosmos Biomedical	CB361550-0000
LSRII flow cytometry	BD Biosciences
Cytocentrifuge	Thermo Scientific
Leica DMR-XA microscope	Leica Microsystems
Cellometer Auto T4 Cell Viability Counter	Nexcelom Bioscience