Fluorescent Proteins:
Introduction - Quantifying calcium - Definitions - Protocols & Examples
 

Protocols & Examples

Protocols for transfection of YCs expressed in cytosol

HeLa cells seeding:

  • Seed 15.000 cells per dish (1,5 mL) in Mat-Tek P35G-1.5-14-C dishes for 24 h.

Transfection (procedure for 1 dish):

  • Prepare DNA solution: dilute 3 µL FuGENE in 100 µL Opti-MEM and mix thoroughly, add 1 µg of DNA, mix again and incubate 30 min at room temperature.
  • Wash the cells gently with PBS x 3.
  • Add 1400 µL of Opti-MEM.
  • Add the DNA solution prepared previously.
  • Incubated at 37ºC overnight (8-10 h). Important: Opti-MEM does not contain antibiotic!
  • Remove transfection mixture.
  • Wash gently with PBS x 3.
  • Add DMEM high glucose + 10 % FCS.
  • Allow cells to express YCs proteins for 3-6 days (incubate at 37ºC and change medium as usual).

Measurement:

  • Use medium without phenol red or the corresponding buffer for measurements.

 

Fluorescent Dyes

 

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