Coat a 10 cm plate with 0.1% gelatin for 0h 20m 0s

From a 80% confluent 10 cm plate of astrocytes obtained from protocol http://www.protocols.io/view/astrocyte-extraction-from-brain-organoids-261ge364wl47/v2

Add 3 mL of for 0h 5m 0s

300rcf,25°C

Add pTALV-fUBIGW-GCAMP8SIRES-PURO lentivirus 1/100 in to the pellet and resuspend the cells

Incubate 0h 5m 0s

Add to a gelatin coated 10 cm plate with a 9 ml warmed media.

48h 0m 0s observe fluorescence levels to confirm infection

Wait 168h 0m 0s for the GCAMPs expression to reach stable levels

Coat a 2 cm plate with 0.1% gelatin for 0h 20m 0s

Plate 50k astrocytes of infected astrocytes

after 48h 0m 0s change to mature astrocyte media

Before the start of imaging aim for 80% confluency.

Place the plate on a

Equipment

Value	Label
Nikon Eclipse TE2000-U microscope	NAME
Microscope	TYPE
Nikon	BRAND
N/A	SKU
http://www.microscope.healthcare.nikon.com/products/stereomicroscopes-macroscopes/smz25-smz18	LINK

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.