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

Published: November 30, 2023

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 Preparation of RBL (RBL-2H3 mast cell line) culture media 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. Filter the media by using a top-va…

Divulgaciones

The authors have nothing to disclose.

Materials

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

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Citar este artículo
An In Vitro Technique to Visualize Exocytosis of Secretory Granules in Mast Cells. J. Vis. Exp. (Pending Publication), e21872, doi: (2023).

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