Unique Approach for Isolating Rat Bone Marrow Neutrophils with Comparable Capacity

Summary

This research outlines two techniques for isolating abundant neutrophil extracellular traps (NETs) from rat bone marrow. One method combines a commercial neutrophil isolation kit with density gradient centrifugation, while the other employs only density gradient centrifugation. Both approaches yield functional NETs surpassing those from peripheral blood neutrophils.

Abstract

The primary aim of this research was to develop a reliable and efficient approach for isolating neutrophil extracellular traps (NETs) from rat bone marrow. This effort arose due to limitations associated with the traditional method of extracting NETs from peripheral blood, mainly due to the scarcity of available neutrophils for isolation. The study revealed two distinct methodologies for obtaining rat neutrophils from bone marrow: a streamlined one-step procedure that yielded satisfactory purification levels, and a more time-intensive two-step process that exhibited enhanced purification efficiency. Importantly, both techniques yielded a substantial quantity of viable neutrophils, ranging between 50 to 100 million per rat. This efficiency mirrored the results obtained from isolating neutrophils from both human and murine sources. Significantly, neutrophils derived from rat bone marrow exhibited comparable abilities to secrete NETs when compared with neutrophils obtained from peripheral blood. However, the bone marrow-based method consistently produced notably larger quantities of both neutrophils and NETs. This approach demonstrated the potential to obtain significantly greater amounts of these cellular components for further downstream applications. Notably, these isolated NETs and neutrophils hold promise for a range of applications, spanning the realms of inflammation, infection, and autoimmune diseases.

Introduction

Neutrophils constitute a critical subset of leukocytes that play a pivotal role in the innate immune response. They are characterized by multilobed nuclei and granules containing various proteases and antimicrobial peptides¹. Neutrophils primarily function through degranulation, phagocytosis, and the formation of NETs. The observation of NETs was first made by Takei et al. in 1996 during an experiment where neutrophils were stimulated with phorbol myristate acetate (PMA)². Subsequently, the process of NET formation was coined "NETosis" by Brinkmann et al.³ in 2004. Their research further i....

Protocol

The study was performed under a project license (No. 20211404A) granted by the Animal Ethics Committee of West China Hospital, Sichuan University, in compliance with the guidelines of the Animal Ethics Committee of West China Hospital, Sichuan University for the care and use of animals. In accordance with ethical guidelines, the rats used in this study were maintained in a controlled environment with a 12 h light/dark cycle, temperature at 22-24 °C and humidity of 50%-60%. The rats were given access to food and wate.......

Discussion

The isolation of neutrophils constitutes a pivotal step in studying NETosis, where the selection of an appropriate isolation method is paramount for obtaining dependable results. An important factor to weigh is the occurrence of lymphocyte contamination during isolation. Addressing this challenge is particularly significant when isolating rat neutrophils from bone marrow. Despite the distinct density range of neutrophils (1.0814-1.0919, with a peak at 1.0919) compared to lymphocytes (1.0337-1.0765, with a peak at 1.0526).......

Materials

Name	Company	Catalog Number	Comments
A488-conjugated donkey antirabbit IgG(H + L)	Invitrogen, USA	A32790
A594-conjugated donkey anti-mouse IgG(H + L)	Invitrogen, USA	A32744
A594-conjugated goat anti-Mouse IgG1	Invitrogen, USA	A21125
Anti-rat myeloperoxidase	Abcam, England	ab134132
Anti-rat neutrophil elastase	Abcam, England	ab21595
Celigo Image Cytometer	Nexelom, USA	200-BFFL-5C
DNase I	Sigma, USA	10104159001
fetal bovine serum (FBS)	Gibco, USA	10099141C
Hank’s Balanced Salt Solution (HBSS)	Gibco, USA	C14175500BT
Hoechst	Thermofisher, USA	33342
Isoflurane	RWD, China	R510-22-10
Mowiol	Sigma, USA	81381
Normal Donkey Serum	Solarbio, China	SL050
Paraformaldehyde	biosharp, China	BL539A
Penicillin-streptomycin	Hyclone, USA	SV30010
Percoll	GE, USA	P8370-1L
Phorbol 12-myristate 13-acetate (PMA)	Sigma, USA	P1585
Picogreen dsDNA Assay Kit	Invitrogen, USA	P11496
Rat neutrophil isolation kit	Solarbio, China	P9200
Red blood cell lysis buffer	Solarbio, China	R1010
Roswell Park Memorial Institute (RPMI) media	Hyclone, USA	SH30809.01B
RWD Universal Animal Anesthesia Machine	RWD, China	R500
Sprague Dawley (SD) rats	Dashuo, China
SytoxGreen	Thermofisher, USA	S7020
Tris-EDTA (TE) buffer	Solarbio, China	T1120
Triton-X-100	Biofroxx, German	1139ML100