Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Summary

The goal of the present protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time analyses of the secretory granule's morphology.

Abstract

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs’ mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules (SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

Introduction

Mast Cells (MC) are immune cells that are best known for their involvement in allergic and inflammatory reactions such as arthritis, asthma, eosinophilic esophagitis, chronic dermatitis and anaphylactic shock ^1,2 as well as other pathologies including coronary artery disease ^3,5 and cancer ^3,4. In addition, MCs play important roles in innate and adaptive immunity, both in host defense against bacteria and parasites and by suppression of immune responses, for example inducing allograft tolerance ^5,6 .

MCs originate from the bone marrow, developing from CD34+/CD117+ pluripotent progenitor cells

Protocol

1. Preparation of RBL Cell Culture Media

1. Mix 500 ml of low glucose Dulbecco's modified Eagle's medium (DMEM) with 56 ml of fetal bovine serum (this makes 10% FBS), and then add 5.5 ml of penicillin streptomycin (this makes ~1% PEST).
2. Filter the media by using 500-1,000 ml Bottle-Top Vacuum Filters with 0.22 μm pore size and store at 4 °C.

2. Culture of RBL Cells

1. Grow RBL cells in a humidified atmosphere of 5% CO₂ at 37 °C in either plates or flasks. RBL cells that are growing in 10 cm plates should be supplemented with 10 ml medium and form confluent monolayer o....

Results

Because of the low transfection efficiency of MCs, genetic manipulations are unlikely to leave an impact on readouts of average secretion measured by endogenous SGs mediators. Nevertheless, by establishing complete co-expression of the reporter gene NPY-mRFP and the co-transfected plasmid at the same cells, monitoring of NPY-mRFP results in monitoring exclusively the cell population that expresses the gene of interest. Therefore, the advantage of this assay compared to conventional methods is the ability to selectively m.......

Discussion

We describe an innovative strategy that combines quantification of MCs exocytosis and four (x, y, z, t) dimension quantifications by time-lapsed three-dimensional imaging of the SGs in living cells using a reporter gene for exocytosis. This technique enables screening of families of proteins for their impact on MC function such as monitoring SGs starting as early as their exit from the Golgi through their maturation, acquisition of exocytosis competence and degranulation. The combination of measurements of exocytosi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Sigma-Aldrich	D6046-500ML	Warm in 37 °C water bath before use
Fetal Bovine Serum	GE health care Life sciences	SH30071.01
Penicillin-Streptomycin	Life technologies
Cellulose acetate membrane, pore size 0.22 μm	Sigma-Aldrich	CLS430769-1EA
Corning tissue-culture treated culture dishes	Sigma-Aldrich	CLS430167
Trypsin/EDTA Solution (TE)	Life technologies	R001100	Warm in 37 °C water bath before use
PIPES dipotassium salt	Sigma-Aldrich	108321-27-3
Calcium acetate hydrate	Sigma-Aldrich	114460-21-8
Magnesium acetate tetrahydrate	Sigma-Aldrich	M5661
L-Glutamic acid potassium salt monohydrate (Potassium glutamate)	Sigma-Aldrich	G1501
4 mm electroporation cuvettes	cell projects	EP-104
GENE PULSER WITH PULSE CONTROLLER & CAPACITANCE	Bio rad
Chambered coverglass	Thermo scientific	155411
24 well, flat bottom	Sigma-Aldrich	CLS3524
Corning 96 well plates	Sigma-Aldrich	CLS3367 or CLS390
96 well plate fluorescence reader- Infinite 200	Tecan
Calcium ionophore A23187	Sigma-Aldrich	C7522	Avoid from direct light exposure
12-O-tetradecanoyl-13-acetate (TPA)	Calbiochem	P3766
anti-DNP monoclonal IgE	Sigma-Aldrich	D8406
DNP-BSA/ DNP-HAS	Sigma-Aldrich	A6661	Avoid from direct light exposure
Triton-x-100	Sigma-Aldrich	T8787
Confocal fluorescent microscope:
Zeiss LSM 510
Leica	SP5
Nikon A1 inverted
Imaris software	BITLANE
Microsoft exel or Prism or other analyses software
Other reagent:
Magnesium Chloride	MERK	5833
Sodium chloride	MERK	6404
Calcium chloride	MERK	2382
Bovine serum albumin	Sigma-Aldrich	A4503
Glucose	BDH Laboratories	284515V
Monosodium phosphate	MERK	5345
Sterile water