Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

Summary

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1⁺ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1⁺ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

Abstract

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer^1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens⁷. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to naïve counterparts^7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression^11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.

Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to naïve MDSC ¹³. MDSC percentages are significantly less in lymphoid compartments of naïve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1⁺ leukocytes from naïve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1⁺ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1⁺ leukocytes from spleens of naïve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas naïve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from naïve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1⁺ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1⁺ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (naïve vs. tumor- bearing) in in vivo and in vitro assays.

Protocol

Prior to starting, prepare the following solutions:

3% Staining Media (SM):

-3% Fetal Bovine Serum (FBS) in 1X Phosphate Buffer Saline (PBS)

MACS Buffer (MB):

- 0.5% Albumin from Bovine Serum (BSA) in 1XPBS

1. Harvest Spleens from Mice

1. Subcutaneously inject 6-8 week of age C57BL/6 mice (Harlan) with 1.5 x 10⁵ murine Panc02 cells suspended in 100 μl 1x PBS (tumor-bearing; TB). Control mice (Naïve) receive 100 μl 1XPBS.
2. Approximately 4 weeks post injection, euthanize mi....

Discussion

This is a detailed method for processing and immunophentyping MDSC populations that is applicable to different lymphoid tissues from various animal models. In particular, autoMACS enrichment can be used for the isolation of various leukocyte populations including Gr-1 depletion of splenocytes ⁴, purification of myeloid subsets from splenocytes and lymph nodes ⁵, isolation of bone marrow neutrophils ¹⁴ and purification of CD8⁺ T cells from spleen and lymph nodes ¹⁵. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1X Phosphate Buffered Saline	Thermo Scientific Hyclone	SH30028.02	Ca2+/Mg2+/Phenol Red-free
Albumin from Bovine Serum (BSA)	Sigma-Aldrich	A7906	Let BSA dissolve undisturbed in PBS; Sterile Environment
Fetal Bovine Serum (FBS)	Thermo Scientific Hyclone	SV3001403HI	Heat Inactivated; Sterile Environment
Rat anti-mouse CD16/32 monoclonal antibody (Fc Block)	BD Biosciences	553142	Sterile Environment
Anti-mouse CD11b (Mac-1) FITC	eBioscience	11-0112	Sterile Environment
Anti-mouse Ly6G (Gr-1) APC	eBioscience	17-5931	Sterile Environment
Anti-mouse Ly6G (Gr-1) PE	eBioscience	12-5931	Sterile Environment
DAPI	Invitrogen	D1306	Serial Dilution Sterile Environment
Cell Dissociation Sieve	Sigma-Aldrich	CD1-1KT	Autoclave before use
70-μm strainer	BD Biosciences	352350	Sterile Environment
1X RBC Lysis Buffer	eBioscience	00-4333-57	Warm to room temperature before use; Sterile Environment
Petri dishes	Fisher Scientific	08-757-12	Sterile Environment
50ml conical tubes	Thermo Fisher Scientific, Inc.	339652	Sterile Environment
5ml 12X75mm polystyrene round bottom tubes	BD Biosciences	352054	Known as FACS tubes; Sterile Environment
96-well V-bottom plates	Corning	3897	Sterile Environment
Trypan Blue	Cellgro	25-900-CI	Sterile Environment
PE MicroBeads	Miltenyi Biotec	130-048-801	Sterile Environment
AutoMACS Pro Separator	Miltenyi Biotec	130-092-545
AutoMACS Columns	Miltenyi Biotec	130-021-101
AutoMACS Running Buffer	Miltenyi Biotec	130-091-221