Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method

Summary

Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method.

Abstract

With a decrease of bone mineral density, bones are more likely to fracture, thus negatively affecting a patient's quality of life. The growth and development of bones are mainly regulated by osteoblasts and osteoclasts. It has been widely accepted that osteoclasts are derived from bone marrow monocyte-macrophage cells (BMMs). BMMs and other hematopoietic stem cells are located in the bone marrow cavity. Therefore, isolating single stable BMMs from different and heterogeneous cell populations is a huge challenge. Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method. Adherent cells cultured for 24-48 h in primary culture were collected. Flow cytometric analysis showed that approximately 37.94% of the cells were CD11b/c+ (monocyte-macrophage surface antigen). Tartrate resistant acid phosphatase (TRAP) staining and western blot analysis demonstrated that BMMs could differentiate into osteoclasts in vitro. The above findings suggested that the secondary adherence cells could be considered as a suitable cellular model for osteoclast differentiation research.

Introduction

It has been reported that monocyte-macrophage lineage cells existing in the bone marrow can differentiate into blood monocytes and tissue macrophages^1,2. The above cells, which can differentiate into osteoclasts to balance bone growth and development, are commonly used as a cell model to induce osteoclasts in vivo^3,4. Bone marrow is a special tissue containing several different types of cells, which include but are not only limited to bone marrow mesenchymal stem cells, bone marrow monocyte-macrophage cells (BMMs), hemato....

Protocol

All experiments in this study were conducted in accordance with the animal experiment guidelines of the Zhejiang Chinese Medical University Laboratory Animal Research Center (Approval No: IACUC-20181029-11).

1. Cell extraction

1. Put the Sprague-Dawley rats (SD rats, 1-10 days old, male or female) in the euthanasia cages filled with CO₂ at a balanced rate of 30%-70% container volume/minute. After the rats lose consciousness (20-60 min), euthanize the rat by.......

Discussion

Osteoclasts are one of the most significant cell types involved in the occurrence and development of bone diseases, as well as one of the primary objects of bone disease research²⁰. Monocyte/macrophages can differentiate into osteoclasts. Since mononuclear macrophages (RAW264.7 cells) are too expensive to buy and are easily activated during culture, it is difficult to perform in vitro differentiation experiments using this cell line. Although several methods have been developed for extrac.......

Materials

Name	Company	Catalog Number	Comments
35 mm2 cell climbing slices	NEST Biotechnology	80102
Anti-cathepsin K	Abcam	ab19027	1:1,000
Anti-CD11 isotype control	Abcam	ab172730	1 μg/test,1.675 mg/Ml
Anti-CD11b/c	Absin	abs124232	1μg/test, 1 mg/mL
Anti-TRAP	Abcam	ab191406	1:1,000
Anti-β-actin	Beyotime	AF5003	1:1,000
Cell climbing slices	NEST Biotechnology	80102
Cell culture dish	corning	430167
Cell culture flask	corning	430168
Dulbecco's modified eagle medium (DMEM)	Gibco	C11995500BT
Fetal bovine serum (FBS)	Gibco	10099141C
Goat anti-rabbit IgG	Abcam	ab150077	for IF, 1:2,000
goat anti-rabbit IgG	Abcam	ab6721	for WB, 1:2,000
M-CSF	Pepro tech	400-28
PBS	Biosharp	BL302A
RANKL	Pepro tech	400-30
SD rat	Shanghai SLAC Laboratory Animal Co, Ltd		1-10 days old
SDS-PAGE gel preparation kit	Solarbio	P1200
TRAP/ALP Staining Kit	Wako	294-67001
Trypsin-EDTA solution	Biosharp	BL512A
Wright-Giemsa solution	Keygen Biotech	KGA225-1