May 23, 2022
Microglia isolation from mouse and culture (modified from UCSD)
- 1California Institute of Technology
External link: https://doi.org/10.7554/eLife.81453
Protocol Citation: rabdelha 2022. Microglia isolation from mouse and culture (modified from UCSD). protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3p7bel25/v1
Manuscript citation:
Abdel-Haq R, Schlachetzki JC, Boktor JC, Cantu-Jungles TM, Thron T, Zhang M, Bostick JW, Khazaei T, Chilakala S, Morais LH, Humphrey G, Keshavarzian A, Katz JE, Thomson M, Knight R, Gradinaru V, Hamaker BR, Glass CK, Mazmanian SK, A prebiotic diet modulates microglial states and motor deficits in α-synuclein overexpressing mice. eLife doi: 10.7554/eLife.81453
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: May 13, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 62520
Keywords: Microglia isolation, UCSD, mouse, ASAPCRN
Abstract
This protocol details microglia isolation from mouse and culture (modified from UCSD).
Attachments
438-926.docx
98KB
Guidelines
General notes
- (Maximum 6 mice per preparation; protocol described below for 6 brains; ~300,000 cells per 7/week old).
- For high yield better use really young mice, let’s say with P7-14 you may get a lot more than above!
- Mechanical dissociation >> enzymatic dissociation.
- Keep everything at 4 °C .
- In vitro: 150k cells/ well (one brain/well) in a 48 well plate).
- Will get much more microglia with pups à use microglia from pups to test antibodies and for general practice.
Materials
HBSS (1x)Gibco - Thermo FisherCatalog #14175-095
2 ml polytetrafluoroethylene pestle (Wheaton)Raptor suppliesCatalog #358026
Corning™ Falcon™Round-Bottom Polypropylene TubesFischer ScientificCatalog #352059
Falcon® 5 mL Round Bottom High Clarity PP Test Tube with Snap Cap SterileCorningCatalog #352063
2 ml glass mortar (Wheaton)Raptor suppliesCatalog #358004
Cell strainer 70um filterFalconCatalog #352350 .
Percoll®Sigma AldrichCatalog #P4937 .
Microglia staining
Please refer staining protocol.
For culture experiments:
Use MG Cell Culture Media supplemented daily IL-34
| A | B | |
| Day0 | 40ng/mL IL34 | |
| After Day0 | 20ng/mL IL34 |
No media change necessary.
Staining buffer:
| A | B | |
| HBSS | 1X | |
| BSA | 1% | |
| EDTA | 1mM |
| A | B | |
| HBSS | 500 ml | |
| BSA | 5 g | |
| EDTA | 1 ml (0.5M EDTA stock) |
Percoll
Isotonic Percoll
| A | B | |
| Percoll | 45ml | |
| 10X HBSS | 5 ml |
e.g. 6 brains:
37% Percoll
| A | B | |
| IsoPercoll | 13.32ml | |
| 1X HBSS | 22.68 ml |
70% Percoll
| A | B | |
| IsoPercoll | 23.1 ml | |
| 1X HBSS | 9.9 ml |
MG Cell Culture Media
| A | B | |
| DMEM/F12 | ||
| FCS | 5% | |
| Anti-anti |
Note
Microglia need approx. 6 days to recover in vitro after isolation before you start experiment.
Microglia isolation from mouse and culture (modified from UCSD)
Microglia isolation from mouse and culture (modified from UCSD)
1h 32m
1h 32m
Deeply anaesthetize with CO2.
Can also use euthanasia.
Perfuse intracardially with ice-cold PBS (~10 to 20ml, liver gets white).
Remove whole brain and place into staining buffer (HBSS 1X – Life Technologies, 14175-095; 1% BSA, 1 millimolar (mM) EDTA) On ice .
Place in 15 ml falcon tube.
Dissect brain into 6 pieces.
Place brain into mini cell culture dish with HBSS buffer.
Remove spinal cord completely.
Cut with dissecting blade.
- Remove cerebellum
- Cut forebrain into several pieces
Put brain pieces into 15ml falcon with HBSS.
Gently homogenize in staining buffer (~10 mL ) using 2 mL polytetrafluoroethylene pestle (Wheaton, 358026), for six brains (8-week-old) polypropylene round-bottom tube (Corning, 352059).
Put brain into clear round bottom test tube (15 ml).
Gently go up and down with pestle a don’t press down on bottom of tube.
Keep tubes On ice .
Homogenize for roughly 00:30:00 .
30m
When supernatant is cloudy, spin briefly to 48 g , store supernatant in collection tube.
Acceleration=5
Deceleration=5
Wait until centrifuge gets to 48 and then immediately stop the spinning.
Put supernatant into a separate collection tube (label tube); 15 ml falcon tube.
Add HBSS + EDTA+BSA buffer into tube with brain chunks.
- Then continue to homogenize the brain chunks in 15 ml tubes (Corning, 352063).
Homogenize for roughly 00:20:00 .
20m
Spin briefly to 48 g when supernatant is cloudy roughly 00:30:00 each time.
30m
Transfer supernatant to collection tube.
Put brain chunks into a smaller tube (corning 352063).
Add buffer to brain chunks and repeat cycle until procedure becomes inefficient (You should have whitish stuff and supernatant is clear) Should have between 15 and 25 ml whitish to yellowish supernatant per brain.
Supernatant should become clear towards the end of dissociation (although there will still be white chunks of microglia left).
Lastly, transfer supernatant in 2 ml glass mortar (Wheaton, 358004), do once up & down with cell suspension SLOWLY.
Step is to remove and separate chunks of microglia.
Add 1.6-1.8 of supernatant into glass mortar.
Use pestle to push down gently into liquid and then remove pestle and transfer supernatant to a new collection tube (50 ml falcon tube).
Keep adding 2 mL of the supernatant from the collection tube to the glass mortar and repeat this process for remaining supernatant.
Filter homogenate onto 70 µm cell strainer (BD Falcon 352350).
Fill homogenate to 40 mL with HBSS + EDTA +BSA in the 50 ml falcon tube.
Can switch to a new filter if it gets clogged.
Centrifuge for 00:12:00 at 400 x g at 18 °C (step 5 acceleration, step 3 brakes).
12m
Pour out supernatant.
Prepare Percoll (Sigma, P4937).
45 mL percoll + 5 mL 10x HBSS (for 6 brains).
Resuspend pelleted homogenate in 6 mL (per brain) of 37% isotonic Percoll in 15 ml centrifuge tube.
37% percoll
- 13.3 percoll (from step 13)
- 22.7 1x HBSS
70% percoll
- 23.1 percoll (from step 13)
- 9.9 1x HBSS
Underlay with 5 mL of 70% isotonic Percoll.
Note
*can also use 50 mL Falcon, e.g. when 5 brains pooled. 22 mL 37% isopercoll, underlay with 13 mL 70% isopercol).
Use a 5 mL pipette for underlay.
Move pipette up very slow as you add more volume but make sure you are not too close to interphase.
Centrifuge at 600 x g for 00:40:00 at 16 °C -18 °C , with “0“ acceleration and deceleration (!).
40m
Remove myelin and everything to about 3 mL above the interphase.
Remove 1-2 ml of debris on top.
Recover cells at the 37%–70% Percoll interphase, this should be ~2.5-3 ml.
First put 2 mL of HBSS+EDTA+BSA into a collection tube (15 ml falcon tube).
Then get interphase (circle around with pipette).
Then get 2-3 ml above the interphase (don't collect liquid below interphase!).
Remove 2 mL of this separated cells into another tube for unstained control cells for FACS.
Centrifuge for 00:10:00 at 400 x g in HBSS 1X/MG media (1:1), remove supernatant.
10m
Fill tube with HBSS before spinning.
Repeat in 14 mL Falcon tube in HBSS once or twice.
Resuspend in 300 µL of HBSS +EDTA + BSA.
Store cells in fridge for staining.