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…
Divulgaciones
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