An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Summary

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Abstract

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8α^Pos and CD8α^Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8α^Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8α^Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8α^Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Introduction

Dendritic cells (DC) were discovered almost forty years ago as the "large stellate (Greek dendron) cell" found in lymphoid organs¹. Many studies have shown that DCs are the only antigen presenting cells that can effectively stimulate naïve T cells². A major function of these cells is the uptake and presentation of antigens and their efficient processing and loading of these onto antigen presenting molecules. In mouse spleen, DCs can be separated into plasmacytoid and conventional subsets. The plasmacytoid DCs are characterized by low expression of CD11c and high levels of B220 and Gr-1. They are also positive for the surface....

Protocol

Animal experiments are done in accordance with approved guidelines from the institutional animal care and usage committee (IACUC). All procedures requiring sterility are performed in a biosafety cabinet.

1. Implantation of B16.Flt3L Melanoma in Mice

1. Culture the Flt-3 expressing B16 melanoma cell line in T25 tissue culture flasks at a density of 0.5 x 10⁶ cells/ml in complete DMEM medium with 10% CO₂ at 37 °C. Grow until the cells reach ~90% confluence (~20 hr).
2. Harvest the cells by trypsin digestion. Aspirate cell culture media and wash the adherent cells with PBS. Add 5 ml of 0.05%....

Results

The outcome of this procedure relies on the expansion of DC subsets by murine Flt-3L expressed by the implanted melanoma cells. The B16.Flt3L tumor was derived from a C57BL/6 mouse, and should be implanted into animals with that strain background in order to avoid failure of the tumor to become established due to rejection. In some cases, it may be desirable to use genetically modified mice to derive DCs with known defects in signaling pathways or receptors of interest. It is important to.......

Discussion

Dendritic cells are accepted to be the major professional antigen presenting cells involved in the priming of T cell responses. Their main function is to survey the tissue microenvironment by taking up and processing antigens for presentation to T cells. In order to study the function of specific DC subsets, these need to be isolated in sufficient numbers using an approach that maintains their normal phenotype and functions. Most protocols rely on the isolation of specific DC subset from naïve mice. Since these cell.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05% Trypsin-EDTA	Life Technologies, Gibco	25300-054
Isoflurane	Sigma-Aldrich	CDS019936-250MG
Collagenase D	Roche Diagnostics	11088858001
DNase I (dry powder)	QIAGEN	79254
200 proof ethanol	Thermo Fisher Scientific	9-6705-004-220	Used to prepare 70% ethanol
RBC lysis buffer	Sigma-Aldrich	R7757
RPMI-1640 medium with L-glutamine	Life Technologies, Gibco	11875-119
DMEM medium with L-glutamine	Life Technologies, Gibco	11995-073
200 mM L-glutamine	Life Technologies	25030081
MEM non-essential amino acids	Life Technologies, Gibco	11140-050
MEM essential amino acids	Life Technologies	11130-051
β-mercaptoethanol	Life Technologies, Invitrogen	21985-023
Sodium pyruvate	Life Technologies	11360-070
HEPES	Life Technologies, Invitrogen	15630
Phosphate buffered saline (PBS Ca2+ and Mg2+ free pH 7.2)	Life Technologies, Invitrogen	20012-050
Dulbecco’s PBS (DPBS with Ca2+ and Mg2+)	Life Technologies, Gibco	14040-182
0.5 M Ethylenediaminetetraacetate (EDTA) solution	Life Technologies	15575-020
Bovine serum albumin (BSA)	Sigma-Aldrich	A2153
Fetal calf serum	Atlanta Biologicals	S11050
Penicillin/streptomycin	Life Technologies, Invitrogen	15140-163
Trypan blue (dry powder)	Sigma-Aldrich	T6146-5G	Prepare 0.08% with PBS
Plasmacytoid Dendritic Cell Isolation Kit II, mouse	Miltenyi Biotech	130-092-786
Magnetic beads conjugated with anti-mouse CD11c	Miltenyi Biotech	130-152-001
CD8αPos mouse DC isolation kit	Miltenyi Biotech	130-091-169
Fc-gamma receptor blocking antibody (Clone 2.4G2)	BD Biosciences	553141
Anti-mouse CD11c-FITC	BD Biosciences	553801
Anti-mouse CD8α-PerCP	BD Biosciences	553036
Anti-mouse B220-PE	BD Biosciences	553090
1 ml syringes	BD	26048
23 G1 needle	BD	305145
100 mm Petri dishes	Thermo Fisher Scientific	875712
Surgical instruments	Kent Scientific	INSMOUSEKIT
Cell strainer (70 µm)	BD	352350
Large Petri plates	Thermo Fisher Scientific	FB0875712
Vacuum filtration system (500 ml (0.22 µm))	Corning	431097
LS columns	Miltenyi	130-042-401
Magnetic stand MACS separator	Miltenyi Biotec	130-042-302
Wide-bore 200 μl pipette tips	PerkinElmer	111623
Corning ultra-low attachment 96-well plates	Corning	CLS3474-24EA
6-8 week old female C57BL/6 mice	Jackson Research Laboratories, Jax
Murine B16.Flt3L melanoma cell line			described by Mach et al., 2000
Serum free DMEM and RPMI media 500 ml DMEM with L-glutamine, or 500 ml RPMI-1640 with L-glutamine 5 ml MEM nonessential amino acids (100x, 10 mM) 5 ml HEPES Buffer (1 M) 5 ml L-glutamine (200 mM) 0.5 ml 2-mercaptoethanol (5.5 x 10-2 M)			Mix all the ingredients in a biosafety cabinet with either DMEM or RPMI media depending on your need. Filter sterilize the media by passing through a 0.22 µm vacuum filtration system.
Complete RPMI and DMEM media			Add 50 ml of heat inactivated fetal calf serum to serum free RPMI or DMEM media to obtain complete media.
MACS buffer			Add 2 ml of 0.5 M EDTA and 10 ml of heat inactivated fetal calf serum to 500 ml of Phosphate-buffered saline (PBS, Ca2+ and Mg2+ Free, pH 7.2) and 2 µg/ml 2.4G2 (Fc-Block antibody).
FACS staining buffer			Dissolve sodium azide to 0.05% (0.25 g per 500 ml) in MACS buffer to obtain FACS staining buffer
10x Collagenase D and DNase 1 stock solution Dissolve 1 g of collagenase D and 0.2 ml of DNase 1 stock (1 mg/ml, 100x) in 20 ml of PBS containing Ca2+ and Mg2+ to obtain a solution of approximately 1,000-2,000 Units of collagenase activity per ml.			This 10x stock solution can be prepared ahead of time and stored at -20 °C for several weeks. Dilute 1 ml of this with 9 ml of serum free RPMI immediately before use.