Obtaining Cell Suspensions from Ischemic Mouse Brain Tissues

Abstract

Source: Pösel, C., et al. Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain. J. Vis. Exp. (2016)

This video demonstrates the procedure for isolating brain cells and infiltrated immune cells from ischemic mouse brain tissues by enzymatic digestion, followed by the removal of cell debris and myelin by density gradient centrifugation, to study inflammation responses in stroke research.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review.

1. Preparation of Reagents

1. Digestion buffer (1 ml per hemisphere): Dissolve a standardized mixture of purified digestive enzymes, e.g., Liberase with low thermolysin concentration (TL) to a concentration of 2U/ml in Hanks Balanced Salt Solution (HBSS) containing calcium (Ca) and magnesium (Mg).
2. Washing buffer with DNAse: Dissolve DNAse I to a concentration of 666U/ml in HBSS (Ca/Mg (calcium/magnesium) free) containing 10% of fetal calf serum (FCS).
3. Washing buffer without DNAse: HBSS (Ca/Mg free) containing 10% of FCS.
4. Density gradient medium (5 ml per hemisphere): prepare stock isotonic density gradient medium (SIP; 100%) by mixing nine parts of density gradient medium with one part of 1.5 M sodium chloride. Dilute SIP to 25% density by adding an appropriate volume of HBSS (Ca/Mg free) containing 3% of FCS.

2. Dissociation of Cerebral Tissue into Single Cell Suspensions

1. Carefully remove brain stem and cerebellum with a clean razor blade. Use a clean razor blade to hemisect the brain and cut each hemisphere along the coronal plane into three pieces of roughly equal size.
2. Mince dissected tissue of each hemisphere through a 100 µm cell strainer using the plunger end of a 5 ml syringe. Continuously rinse the cell strainer with ice-cold HBSS (+Ca/Mg). Store homogenized samples on ice.
3. Centrifuge at 286 x g and 4° C for 5 min, carefully discard the supernatant. Resuspend the pellet in 1 ml of digestion buffer and transfer the suspension into a 2 ml tube. Incubate suspension under slow continuous rotation at 37 °C for 1 hr.
4. Sieve the cell suspension through a 70 µm cell strainer and rinse thoroughly with 3 ml of washing buffer containing DNAse followed by 15 ml of DNAse-free washing buffer. Centrifuge at 286 x g and 18 °C for 5 min and discard the supernatant. Note: The use of DNAse eliminates DNA mucus caused by cell lysis, which might lead to cell aggregation, compromising cell survival.

3. Density Gradient Centrifugation for Removal of Myelin and Cell Debris

1. Resuspend the cell pellet in 5 ml of 25% density gradient medium and pipette the suspension to a 15 ml tube. Mix thoroughly by repeated and gentle pipetting, avoiding bubble formation. Note: Density gradient medium should be used at RT to prevent cell clumping.
2. Centrifuge at 521 x g and 18 °C for 20 min. Use the lowest acceleration profile of the rotor and allow the rotor to stop without a brake. Gently remove the tubes from the centrifuge without shaking. Carefully aspirate the myelin coat and the supernatant while preserving the cell pellet at the bottom of the tube. Note: Make sure to remove the entire myelin coat, as any leftover will impair flow cytometric analysis.
3. Resuspend the pellet in 10 ml of DNAse-free washing buffer and pipette the suspension to a new 15 ml tube. Note: This washing step is crucial since sufficient removal of density gradient medium significantly contributes to the quantity and quality of the final cell sample.
4. Centrifuge again at 286 x g and 10 °C for 5 min and discard the supernatant. Resuspend cells in 100 µl of cold washing buffer and determine cell counts and viability by trypan blue exclusion in a hemocytometer. Store samples at 4 °C and further process them quickly.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MACSmix Tube Rotator	Miltenyi Biotec GmbH
Heraeus Multifuge 3SR+	Thermo Scientific
Hank's buffered salt solution (HBSS)	Gibco/Life Technologies GmbH	14175-129
HBSS with Calcium/Magnesium	Gibco/Life Technologies GmbH	24020-091
Liberase TL	Roche Diagnostics GmbH	5401020001	Use at room temperature
Percoll Plus	GE Healthcare Europe GmbH	17-5445-01
Fetal bovine serum	PAN Biotech	3302-P101003
Trypan blue	Gibco/Life Technologies GmbH	15250-061
Trucount	BD Biosciences	340334
Phosphate buffered saline	Biochrom AG	L 182-10
DNase I	Roche Diagnostics GmbH	11284932001
Silicone Tubing, 1m	World precision instruments	503022
Fine Iris Scissors sharp/blunt	Fine Science Tools	14028-10
Straight 1x2 teeth forceps	Fine Science Tools	11021-14
Blunt-end Forceps	Fine Science Tools	11008-13
5ml syringe plunger	Carl Roth GmbH (Braun)	EP96.1
Cell strainer, 100µm	Dr. I. Schubert, BD	2360-00
Omnican Fine dosage syringe 1ML	Braun	TBD
Cell strainer, 70µm	Greiner Bio-One GmbH	542 070
serological pipettes, 10ml	Greiner Bio-One GmbH	607180
serological pipettes, 10ml	Sarstedt AG&Co	86,12,54,025
serological pipettes, 25ml	Greiner Bio-One GmbH	760180
serological pipettes, 5ml	Greiner Bio-One GmbH	606180
serological pipettes,25ml	Sarstedt AG&Co	86,16,85,020
serological pipettes,5ml	Sarstedt AG&Co	86,12,53,025
Tips, 0,1-10µl	Corning B.V.Life Sciences	4840
Tips, 100-1000µl	Greiner Bio-One GmbH	740290
Tips, 10-200µl	Greiner Bio-One GmbH	739296
Reaction tubes 1,5ml	Greiner Bio-One GmbH	616201
Reaction tubes 2ml	Greiner Bio-One GmbH	623201
Bacteriological petri dish, 94x16mm	Greiner Bio-One GmbH	633180
Falcon 15ml	Greiner Bio-One GmbH	188271
Falcon 50ml	VWR International GmbH (BD)	734-0448
Neubauer hemocytometer	Biochrom AG	PDHC-N01
razor blade	Carl Roth GmbH	CK07.1