Isolation and Activation of Murine Lymphocytes

Summary

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Abstract

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen^1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses³. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting⁴ and the investigation of proliferative abilities by ³H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling^5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Introduction

Mature lymphocytes generally exist in the resting state if there is no pre-existing infection or inflammation in the individual. Therefore, it is important to retain the naïve status of lymphocytes during the isolation process before performing in vitro or in vivo functional assays. The key to ensuring consistent and reproducible results is to limit any unnecessary manipulation of the cells.

Magnetic cell sorting utilizes antibodies and microbeads to label cells so as to enrich the cell population of interest. With this approach, there are two purification strategies: positive enrichment and negative depletion. Posit....

Protocol

All mice are bred and maintained under specific pathogen-free conditions and all mouse protocols are conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee.

1. Preparation of Buffers and Reagents

1. Prepare complete Roswell Park Memorial Institute (RPMI) medium (10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (100 IU/ml)/streptomycin (100 µg/ml), 55 µM 2-mercaptoethanol).
2. Prepare 20x Balanced Salt Solution (BSS) Stock 1 and Stock 2, Separately.
   1. Prepare 20x BSS stock 1 (111 mM dextrose, 8.8 mM potassium phosphate, 26.7 mM sodium phosp....

Results

Magnetic cell purification of lymphocytes allows users to purify a target cell population in a relatively short amount of time. Using our depletion protocol, we were able to increase the percentage of CD8 T cells (OT-I in recombination-activating gene-1 (RAG-1)-deficient mice) from 72.8% (before purification) to 94.2% (after purification; Figure 1A)^4,5. These purified lymphocytes can then be used for downstream functional assays to determine lymphocyte prolifer.......

Discussion

In this protocol, we demonstrate a procedure for purifying lymphocytes from lymphoid organs. Cell purification using magnetic bead sorting is a fast and simple method that yields viable, highly purified target cells.

Critical Steps within the Protocol

Cell viability and cell yield

Maintaining viability of hematopoietic lineage cells in vitro is critical to ensuring successful and reproducible experiments. Chemical .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Materials
RPMI 1640 (without L-Glutamine)	Gibco	31870025
Fetal Bovine Serum			Heat inactivated
L-glutamine	Gibco	25030024
Penicillin/Streptomycin	Gibco	15140114
2-mercaptoethanol	Gibco	21985023
Anti-CD43 magnetic microbeads	Miltenyi Biotec	130-049-801	Mix well prior use
Streptavidin microbeads	Miltenyi Biotec	130-048-101	Mix well prior use
Anti-Annexin V magnetic beads	Miltenyi Biotec	130-090-201	Mix well prior use
MACS LD	Miltenyi Biotec	130-042-901
96-well U-bottom sterile culture plate	Greiner Bio-one	650180
96-well F-bottom sterile culture plate	Greiner Bio-one	655180
100 μm cell strainer mesh			To sterilize using UV radiation prior use
0.2 μm  sterile disposable filter units	Nalgene	567-0020	Can be substituted with any sterile filter device
CellTrace Violet	Invitrogen	C34557	CTV for short; alternative to CFSE
CellTrace Yellow	Invitrogen	C34567	CTY for short; alternative to CFSE
CellTrace Far Red	Invitrogen	C34564	CTFR for short; alternative to CFSE
Cell Proliferation Dye eFluor 670	eBioscience	65-0840	CPD670 for short; alternative to CFSE
PKH26	Sigma Aldrich	PKH26GL	PKH26, alternative to CFSE
Name	Company	Catalog Number	Comments
Chemicals
Dextrose	Sigma Aldrich	G7021
Potassium phosphate monobasic	Sigma Aldrich	P5655
Sodium phosphate dibasic	Sigma Aldrich	S5136
Phenol Red	Sigma Aldrich	P0290
Calcium chloride dihydrate	Sigma Aldrich	C7902
Potassium chloride	Sigma Aldrich	P5405
Sodium chloride	Merck Millipore	S7653	Can use from other sources
Magnesium chloride hexahydrate	Sigma Aldrich	M2393
Magnesium sulfate	Sigma Aldrich	M2643
Ammonium chloride	Sigma Aldrich	A9434
Tris-base
Dimethyl Sulfoxide	Sigma Aldrich	D8418
(5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)	Molecular Probes	C-1157	Reconstitute in DMSO
Phorbol 12,13-dibutyrate (PBDU, Phorbol ester)	Sigma Aldrich	P1269
A23187 (Calcium ionophore)	Sigma Aldrich	C7522
Name	Company	Catalog Number	Comments
Antibodies and recombinant protein
CD11b biotin (clone m1/70)	Biolegend	101204	T cell depletion cocktail
CD11c biotin (clone N418)	Biolegend	117304	T cell depletion cocktail
Gr-1 biotin (clone RB6-8C5)	Biolegend	108404	T cell depletion cocktail
Ter119 biotin (clone Ter119)	Biolegend	116204	T cell depletion cocktail
TCR-γδ biotin (clone GL-3)	Biolegend	118103	T cell depletion cocktail
CD19 biotin (clone 6D5)	Biolegend	115504	T cell depletion cocktail
B220 biotin (clone RA3-6B2)	Biolegend	103204	T cell depletion cocktail
CD49b biotin (clone DX5)	Biolegend	108904	T cell depletion cocktail
CD4 biotin (clone GK1.5)	Biolegend	100404	T cell depletion cocktail
CD8 biotin (clone 53-6.7)	Biolegend	100704	T cell depletion cocktail
F(ab’)2 goat anti-mouse IgM (plate coated)	Jackson ImmunoResearch	115-006-075	50 µl/well for coating (96-well)
Anti-mouse CD40 mAb (plate coated)	Pharmingen	553722	50 µl/well for coating (96-well)
Recombinant IL-4	ProSpec	Cyt-282
LPS from E. coli Serotype 055:B5	Sigma Aldrich	L-4005
Anti-CD3 (clone clone OKT3) (plate coated)	eBioscience	16-0037-85	50 µl/well for coating (96-well)
Anti-CD28 (clone clone 37.51) (plate coated)	eBioscience	16-0281-85	50 µl/well for coating (96-well)
Recombinant IL-2	ProSpec	Cyt-370
Albumin from chicken egg white, Ovalbumin	Sigma Aldrich	A7641