An In Vitro Technique to Visualize Exocytosis of Secretory Granules in Mast Cells

Abstract

Source: Klein, O., et al. Imaging FITC-dextran as a Reporter for Regulated Exocytosis J. Vis. Exp. (2018).

This video demonstrates a technique to visualize exocytosis in mast cells. The cells are incubated with FITC-dextran — a fluorescent reporter — which is taken up by cells inside secretory granules (SGs), where the acidic pH quenches its fluorescence. Upon stimulation of granule exocytosis, the medium's higher pH induces reporter fluorescence inside the fused SGs, which is visualized using a fluorescence microscope.

Protocol

1. Preparations

1. Preparation of RBL (RBL-2H3 mast cell line) culture media
   1. Mix 500 mL of low glucose Dulbecco's modified Eagle's medium (DMEM) with 56 mL of fetal bovine serum (FBS), 5.5 mL of penicillin-streptomycin-nystatin solution, 5.5 mL of L-Glutamine 200 mM solution. This results in low glucose DMEM supplemented with 10% FBS, 100 µg/mL streptomycin, 100 units/mL penicillin, 12 units/mL nystatin, and 2 mM L-glutamine.
   2. Filter the media by using a top-vacuum filter of 0.22 µm pore size and store at 4 °C.
2. Maintenance of RBL cells.
   1. Grow RBL cells to a maximum conflu....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM low glucose	Biological Inductries	01-050-1A
Fetal bovine serum	Gibco	12657
Pen-strep-nystatin solution	Biological Inductries	03-032-1B
L-Glutamine 200 mM solution	Biological Inductries	03-020-1A
FITC dextran 150K	Sigma-Aldrich	46946-500MG-F
Trypsin/EDTA Solution B	Biological Inductries	03-052-1A	Warm in 37 °C water bath before use
Top-vacuum filter of 0.22 µm pore size	Sigma-Aldrich	CLS430769-1EA
Cellulose acetate syringe filter unit, 0.22 µm pore size	Sartorius	16534K
Chambered coverglass	Thermo scientific	155411
Corning tissue-culture treated culture dishes	Sigma-Aldrich	CLS430167
Bovine serum albumin	Sigma-Aldrich	A4503
Anti-DNP monoclonal IgE	Sigma-Aldrich	D8406
DNP-HSA (Ag)	Sigma-Aldrich	A6661	Avoid direct light exposure
Hepes buffer 1M, pH 7.4	Biological Inductries	03-025-1B
CaCl2	MERK	102382
Glucose	BDH Laboratories	284515V
Ammonium chloride	MERK	1145
PIPES dipotassium salt	Sigma-Aldrich	108321-27-3
Calcium acetate hydrate	Sigma-Aldrich	114460-21-8
Magnesium acetate tetrahydrate	Sigma-Aldrich	M5661
Potassium glutamate (L-Glutamic acid potassium salt monohydrate)	Sigma-Aldrich	G1501
NaH2PO4	MERK	6346
NaCl	MERK	106404
MgCl2	MERK	105833
KCl	MERK	104936
Electroporator	BTX	ECM830
Confocal microscope, Zeiss	Zeiss	LSM 5 Pascal, Axiovert 200M	Used in Figure 3. Equipped with an electronic temperature-controlled
airstream incubator
Confocal microscope, Zeiss	Zeiss	LSM 800, Axio Observer.Z1 /7	Used in Figure 2. Equipped with a GaAsP detector an electronic temperature-controlled
airstream incubator
Confocal microscope, Leica	Leica	SP5	Used in Figure 1. Equipped with a leica HyD detector and an top-stage incubator (okolab)
RBL-2H3 cells			RBL-2H3 cells were cloned in the lab of Reuben P. Siraganian. See reference 67