Oct 28, 2022

Public workspaceCalcium imaging in astrocytes

DOI
dx.doi.org/10.17504/protocols.io.36wgqj7wyvk5/v1
  • 1Icahn School of Medicine
gustavo.parfitt Parfitt
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.36wgqj7wyvk5/v1
Protocol Citation: gustavo.parfitt Parfitt 2022. Calcium imaging in astrocytes. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqj7wyvk5/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: October 27, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 71942
Keywords: ASAPCRN
Abstract
Calcium imaging in astrocytes
Protocol materials
ReagentTrypLE™ Select Enzyme (1X), no phenol redThermo FisherCatalog #12563029
Step 3
ReagentAstrocyte MediumScienCellCatalog # #1801
In 2 steps
Coat a 10 cm plate with 0.1% gelatin for Duration00:20:00

20m
From a 80% confluent 10 cm plate of astrocytes obtained from protocol https://www.protocols.io/view/astrocyte-extraction-from-brain-organoids-261ge364wl47/v2
Add 3 mL of ReagentTrypLE™ Select Enzyme (1X), no phenol redThermo FisherCatalog #12563029 for Duration00:05:00

5m
Centrifigation300 rcf, 25°C, 00:03:00

3m
Add pTALV-fUBIGW-GCAMP8SIRES-PURO lentivirus 1/100 in ReagentAstrocyte MediumScienCellCatalog # #1801 to the pellet and resuspend the cells

Incubate Duration00:05:00

5m
Add to a gelatin coated 10 cm plate with a 9 ml ReagentAstrocyte MediumScienCellCatalog # #1801 warmed media.

Duration48:00:00 observe fluorescence levels to confirm infection

2d
Wait Duration168:00:00 for the GCAMPs expression to reach stable levels

1w
Coat a 2 cm plate with 0.1% gelatin for Duration00:20:00
20m
Plate 50k astrocytes of infected astrocytes
after Duration48:00:00 change to mature astrocyte media

2d
Before the start of imaging aim for 80% confluency.
Place the plate on a
Equipment
Nikon Eclipse TE2000-U microscope
NAME
Microscope
TYPE
Nikon
BRAND
N/A
SKU
plate holder.
Continuously perfused with ACSF with the following composition (in mM): NaCl 125, KCl 5, D-Glucose 10, HEPES-Na 10, CaCl2 3.1, MgCl2 1.3. using a ValveBank8 II controller.
Using a 10X objective. GCaMP8s was excited using a 480 nm (Mic-LED-480A, Prizmatix), an HQ480/40x excitation filter, a Q505LP dichroic mirror, and an HQ535/50m emission filter (Semrock).
Sample at a rate of 4.7 fps with a frame exposure of 200 ms at 160x120 pixels (4x4 binning).
ROI segmentation of GCaMP8s, raw fluorescence extraction, and background correction can be performed with Nikon Elements software.