A diacylglycerol photoswitching protocol for studying TRPC channel functions in mammalian cells and tissue slices.

A diacylglycerol photoswitching protocol for studying TRPC channel functions in mammalian cells and tissue slices.

STAR protocols (2021-05-25)

Trese Leinders-Zufall, Ursula Storch, Michael Mederos Y Schnitzler, Navin K Ojha, Kohei Koike, Thomas Gudermann, Frank Zufall

PMID34027485

ABSTRACT

Small molecular probes designed for photopharmacology and opto-chemogenetics are rapidly gaining widespread recognition for investigations of transient receptor potential canonical (TRPC) channels. This protocol describes the use of three photoswitchable diacylglycerol analogs-PhoDAG-1, PhoDAG-3, and OptoDArG-for ultrarapid activation and deactivation of native TRPC2 channels in mouse vomeronasal sensory neurons and olfactory type B cells, as well as heterologously expressed human TRPC6 channels. Photoconversion can be achieved in mammalian tissue slices and enables all-optical stimulation and shutoff of TRPC channels. For complete details on the use and execution of this protocol, please refer to Leinders-Zufall et al. (2018).

MATERIALS

Product Number

Brand

Product Description

P4333

Sigma-Aldrich

Penicillin-Streptomycin, Solution stabilized, with 10,000 units penicillin and 10 mg streptomycin/mL, 0.1 μm filtered, BioReagent, suitable for cell culture

G8877

Sigma-Aldrich

Guanosine 5′-triphosphate sodium salt hydrate, ≥95% (HPLC), powder

D8537

Sigma-Aldrich

Dulbecco′s Phosphate Buffered Saline, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture

03777

Sigma-Aldrich

Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, BioUltra, ≥99.0% (T)

T3924

Sigma-Aldrich

Trypsin-EDTA solution, 1 ×, sterile; sterile-filtered, BioReagent, suitable for cell culture, 0.5 g porcine trypsin and 0.2 g EDTA, 4Na per liter of Hanks′ Balanced Salt Solution with phenol red

70967

Sigma-Aldrich

GeneJuice^® Transfection Reagent, Non-lipid based chemical transfection reagent optimized for maximum transfection efficiency, ease-of-use, and minimal cytotoxicity on a wide variety of mammalian cells.

M4655

Sigma-Aldrich

Minimum Essential Medium Eagle, With Earle′s salts, L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture

A4926

Sigma-Aldrich

1,2-Bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, 98%

H0887

Sigma-Aldrich

HEPES solution, 1 M, pH 7.0-7.6, sterile-filtered, BioReagent, suitable for cell culture