Sep 20, 2022
Calcium Imaging in mDA neurons
- 1UCL Institute of Neurology
Protocol Citation: gurvir.virdi 2022. Calcium Imaging in mDA neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4j7e8lo5/v1
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: September 20, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70289
Keywords: ASAPCRN
Abstract
Calcium imaging using Fura-2, Fluo-4 and X-Rhod-1.
To measure cytosolic calcium, 5 uM Fura-2 or Fluo-4 was added to cells at room temperature for 40 minutes made up in HBSS (Invitrogen).
To measure mitochondrial calcium, cells were incubated with 2μM X-Rhod-1 in HBSS for 40 minutes.
All probes are from Molecular Probes (Thermo Fisher Scientific). Fura-2-AM is a ratiometric dye with a high affinity for Ca2+. It is used to measure [Ca2+]c.
Fluo-4-AM is a calcium indicator.
X-Rhod-1-AM is a calcium indicator which localises to mitochondria.
Cells are then washed 2x in HBSS at room temperature. They are now ready for imaging.
[Ca2+]c is monitored in cells by obtaining the ratio between the excitation at 340 nm (high Ca2+) and 380 nm (low Ca2+) for which fluorescence light was reflected through a 515 nm long pass filter.
Microscopy for Fluo-4:
Live-cell imaging was performed excited by a 488 nm laser and measured at 520 nm. A time-series with 5 second intervals was performed to establish basal fluorescence before 50mM KCl was added to depolarise the membrane and measure fluorescence intensity increase, and recovery.
Microscopy for Fura-2 [Ca2+]c:
The cells were excited sequentially at 340 and 380nm using light from a Xenon arc lamp. A time-series with 1 or 5 second intervals was performed to establish basal fluorescence before 50mM KCl was added to depolarise the membrane.The emitted fluorescence was measured at 515 nm on a cooled camera device (CCD).
[Ca2+]c using Fura-2 AM was also imaged on a Nikon Ti2 inverted microscope with Perfect Focus System, an ASI motorised XY stage with piezo Z and an Okolab environmental chamber with a CO2 mixer. Imaged were acquired using an Andor iXon Ultra897 EMCCD camera. Cells were excited with a Cairn FuraLED light engine optimised for 340 and 380 nm with a dichroic mirror T400lp (Chroma) and an emission filter ET510/80m (Chroma), using a 40x 1.3 NA S Fluor objective. The microscope was controlled with Micro-Manager v2.0
Microscopy for X-Rhod-1:
Live-cell imaging was performed excited by a 561 nm laser. A time-series with 5 second intervals was performed to establish basal fluorescence before 50mM KCl was added to depolarise the membrane and measure fluorescence intensity increase, and recovery giving readouts for mitochondrial calcium.