Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Summary

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Abstract

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils ≥ 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Introduction

Innate immune cells constitute an essential proportion of the cells within the tumor microenvironment and have been associated with tumor malignancy in patients and animal models of cancer¹. Recently, it has become more widely appreciated that chronic immune responses play critical roles in promoting tumor progression, metastasis and resistance to chemotherapies². Macrophages are important innate immune cells that have been shown to directly regulate tumor cell response to chemotherapy ^3,4. However, the role of neutrophils, key players in the innate immune system, in regulating tumor response to anti-cancer treatment is not known. The ....

Protocol

1. Neutrophil Isolation and Co-culture with Primary Leukemic Cells

NOTE: Procedures were conducted under the approval of Lyon Hospital Ethics committee with all patients signing informed consent.

1. Isolation of Primary Leukemic Cells and Neutrophils
   1. Collect tubes of peripheral blood on EDTA (1.8 mg EDTA per milliliter of blood) from patients diagnosed with chronic lymphocytic leukemia (CLL).
   2. Add each 15 ml of blood to a sterile 50 ml tube and dilute with 15 ml RPMI (dilution 1:1), then carefully and slowly add 15 ml of density gradient solution to the bottom of the tube without mixing the ph....

Results

Density gradient separation method described here provides primary leukemic cells and unstimulated neutrophils isolated from the blood of CLL patients. Figure 1A represents the different blood layers obtained after density gradient centrifugation (from top to bottom: platelets and plasma, white ring represents the mononuclear cells, density gradient solution, granulocytes and erythrocytes). Figure 1B and 1C show the differences in the morphological appear.......

Discussion

Described here, an effective, simple, fast and inexpensive protocol for the isolation of neutrophils from human blood with high purity using density gradient centrifugation approach and within the same step mononuclear cells are also separated and recovered. The isolated cell populations are ≥90% pure.

Several methods are available for neutrophil isolation from human blood. These include similar methods using discontinuous gradients^11,12, or using commercial kits for neutrophi.......

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
N-acetyl-L-alanyl-L-glutamine (L-Glutamine)	Life technologies	25030-024
Penicillin streptomycin (Pen Strep)	Life technologies	15140-122
Bruton's tyrosine kinase (Btk) inhibitor (Ibrutinib)	CliniSciences	A3001
Vincristine	EG labo
BD Matrigel basement membrane matrix	BD Biosciences	354234	Put at 4 oC overnight (O/N) before the day of the experiment
Red cell lysis buffer	BD Biosciences	555899
Ficoll (Pancoll)	PAN Biotech	P04-60500
Dextran	Sigma-Aldrich	D8906
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D8418
Retinoic acid	Sigma-Aldrich	R2625
Bovine serum albumin (BSA)	Sigma-Aldrich	A7906
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich	E5134
Sodium chloride (NaCl)	Euromedex	S3014
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