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