Mar 04, 2025
Primary Astrocyte Culture
- Emily Tabaie1
- 1University of California, Riverside
Protocol Citation: Emily Tabaie 2025. Primary Astrocyte Culture. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7moj3gwz/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: March 03, 2025
Last Modified: March 04, 2025
Protocol Integer ID: 123707
Abstract
This protocol is for the isolation and preparation of primary cortical astrocytes cultures from the pups of C57BL/6 mice.
Primary Astrocyte Culture
Primary Astrocyte Culture
Day 1: Take 6 newborn mice (1-3 days postpartum) back to lab
Dissect out the brain and place in a 50 mL conical that has 10 mL of cold washing media, and keep on ice
Repeat steps 2 until all 6 brains are out and pool into the same conical
Take a new 50 mL conical and place a 40 um strainer inside it
Dump all 10 mL of cold washing media plus the brains into the strainer and smash the brains through the strainer with the plunger of a 5 mL syringe
Wash the filter with 10 mL of cold washing media
Spin samples at 2,000 rpm for 10 minutes at 4°C
Decant media and wash with 20 mL of cold washing media
Spin samples at 2,000 rpm for 10 minutes at 4°C
Repeat steps 9 and 10
Decant media and resuspend samples in 36 mL of pre-warmed complete astrocyte media
Plate 6 mL of cell mixture to a T-25 flask keeping the cap loose
Day 3: Dump out media and add 8 mL of pre-warmed completed astrocyte media
Day 5: Dump out and add 8 mL of pre-warmed completed astrocyte media
Day 7: Dump out and add 8 mL of pre-warmed completed astrocyte media
Day 8: Shake flasks for 1.5 to 2 hours at 260 rpm at 37°C
Take off the media and add 6 mL of pre-warmed complete astrocyte media
Shake flasks at 100 rpm for 24 hours at 37°C
Day 9: Aspirate the media with dislodged cells and wash with 5 mL of pre-warmed HBSS
Aspirate the HBSS
Add 1-2 mL of pre-warmed Trypsin EDTA and leave in the 37°C incubator until cells are lifted
Add 5 mL of pre-warmed complete astrocyte media to stop the reaction
Pipette all the flasks in to one 50mL conical tube and centrifuge at 1,200 rpm for 5 minutes at 20°C
Dump out the supernatant and resuspend the pellet in 20 mL of pre-warmed complete astrocyte media
Count cells using automatic cell counter and resuspend the cells to 1x10^6 - 3x10^6 cells/mL for plating
Plate 1x10^6 cells/mL per T-75 vented flask with 20 mL of pre-warmed media
When cells are 100% confluent, parafilm the vented caps and can store cells in mammalian incubator for up to 2 months