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

Published: March 29, 2024

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 Differentiation of Human Promyelocytic (HL60) Cells into Neutrophil-like Cells 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</…

Representative 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 labeled with anti-CD11b-AF700 or anti-CD38-APC followed by flow cytometry analysis. After gating on each cell popul…

Divulgations

The authors have nothing to disclose.

Materials

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

Tags

Play Video

Citer Cet Article
Determining the Role of Neutrophil-Like Cells in Lymphoma Cell Sensitivity to a Test Drug. J. Vis. Exp. (Pending Publication), e22011, doi: (2024).

View Video