Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

Summary

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

Abstract

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (>10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 10⁷ CD11c⁺ cells (DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Introduction

Dendritic cells (DCs) are the most powerful antigen-presenting cells (APCs) for activating naïve T cells and inducing specific cytotoxic T lymphocyte (CTL) responses against infectious diseases, allergy diseases, and tumor cells^1,2,3. DCs are the primary link between innate immunity and adaptive immunity and play an essential role in immunological defense and the maintenance of immune tolerance. In the last 40 years, many researchers have sought to define the subsets of DCs and their functions in inflammation and immunity. As per those studies, DCs develop along the myel....

Protocol

All procedures were approved by the Nanjing Medical University Animal Care and Use Committee.

1. Isolation of bone marrow and preparation of BM cells

1. Sacrifice C57BL/6 mice (18-22 g, 6-8 weeks old) via CO₂ asphyxiation. Fix the mouse on the mouse operating table. Disinfect the surfaces with 70% ethanol.
2. Cut the skin of the leg to expose the muscles and femoral artery. Clamp and tear off the femoral artery using two forceps, then pull the proximal end toward the abdomen.
   NOTE: Do not cut the femoral artery directly. Otherwise, it will cause excessive bleeding and contaminate the field ....

Results

The 1 x 10⁷-1.7 x 10⁷ cells were extracted from two femurs and were re-suspended in 24 mL of medium before being planted in a 6-well plate (Figure 1A). After 2 days, non-adherent cells were removed by completely changing the culture medium. Before changing the medium, a significant number of suspended cells were observed (Figure 1B). After 3 days of culture, small cell colonies began to form. On the sixth day, the size and number of colonie.......

Discussion

Humans and mice have different DC subsets, including classical DCs (cDCs, including cDC1s and cDC2s) plasmacytoid DCs (pDCs), and monocyte-derived DCs (MoDCs)^9,10,11. It is generally accepted that cDC1s regulate cytotoxic T lymphocyte (CTL) responses to intracellular pathogens and cancer, and cDC2s regulate immune responses to extracellular pathogens, parasites, and allergens¹². A significant number of DC.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
β-Mercaptoethanol	Solarbio	M8211
6-well plate	Corning	3516
APC-MHC II	Biolegend	116417
FBS	Gibco	10100
PE-CD80	Biolegend	104707
Penicillin-Streptomycin	Solarbio	P1400
Percp/cy5.5-CD11c	Biolegend	117327
PRMI-1640	Thermo	11875093
Recombinant Mouse GM-CSF	Solarbio	P00184
Recombinant Mouse IL-4	Solarbio	P00196
TruStain Fc PLUS (anti-mouse CD16/32) Antibody	Biolegend	156603