Feb 28, 2023

Public workspaceCalcium fluorimetry with the FLIPR Calcium 6 kit on FlexStation 3 V.2

PLOS One
DOI
dx.doi.org/10.17504/protocols.io.3byl4jdmjlo5/v2
  • Angus Li1,
  • Samuel Liu1,
  • Rennica Huang2,
  • Seungkirl Ahn1,
  • Robert J Lefkowitz1,2,3
  • 1Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America;
  • 2Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, United States of America;
  • 3Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina, United States of America
Angus Li
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DOI: dx.doi.org/10.17504/protocols.io.3byl4jdmjlo5/v2
Protocol Citation: Angus Li, Samuel Liu, Rennica Huang, Seungkirl Ahn, Robert J Lefkowitz 2023. Calcium fluorimetry with the FLIPR Calcium 6 kit on FlexStation 3. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4jdmjlo5/v2Version created by Angus Li
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 28, 2023
Last Modified: February 28, 2023
Protocol Integer ID: 77743
Funders Acknowledgement:
Howard Hughes Medical Institute
National Institutes of Health
Grant ID: R01 HL16037
Abstract
This protocol details an experimental procedure used to generate results described in the manuscript Li, A., Liu, S., Huang, R., Ahn, S., & Lefkowitz, R. J. (2023). Loss of biased signaling at a G protein-coupled receptor in overexpressed systems.
Plate U2OS-TetOn-AT1R in microplates (Black with clear bottom, lysine- coated Corning 3842) at 15000 cells/well 2 days prior to assay
Add doxycycline and optionally PTX to wells 14 hours before replacement with loading buffer
Remove one vial of Calcium 6 Assay Reagent (Component A, good for 2 plates) from the freezer and equilibrate to room temperature.
Dissolve contents of one Component A vial by adding 10 ml of 1X HBSS Buffer plus 20 mM HEPES. Mix by vortexing (~1-2 min) until contents of vial are dissolved. It is important that contents are completely dissolved to ensure reproducibility between experiments.
Remove cell plates from the incubator, Calcium 6 Kit - add an equal volume of loading buffer to each well in the dark (i.e. 120ul HBSS+HEPES + 40ul of Dye/well (4X) for a 96-well plate in the original protocol). Use 11.8ml of HBSS+HEPES +4.8ml of Dye (4X) for one plate. Save the dissolved dye solution in -20.
Return plates to the incubator and incubate two hours at 37 C, 5% CO2
Optionally, add YM-254890 1 hour prior to reading
30 minutes before experiment (1.5 hours into incubation), prepare the reagents (5X, 40ul/well+20ul volume). The ligand needs to be plated in a 96 well with round bottom (The FlexStation will add ligand, shake, and measure sequentially).
Turn on FlexStation 30 minutes before experiment and warm up to 37.
Place tips and ligand plate
After the two-hour incubation period, transfer the assay plate directly to the FlexStation instrument assay plate carriage 10 minutes prior to reading to re-equilibrate temperature, and then run the assay.
In an individual well or column of wells, the calcium flux peak(s) should be complete within 1 to 3 minutes after addition. For an entire plate however, the protocol will not complete until all chosen columns are finished. 40-45 min for entire 96 well plate.