Isolating Tumor-Infiltrating Immune Cells from a Murine Brain

Abstract

Source: Baker, G. J., et al., Lowenstein, P. R. Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells. J. Vis. Exp. (2015)

The video demonstrates the isolation of tumor-infiltrating peripheral blood mononuclear cells from a mouse brain using mechanical dissociation and enzymatic digestion. This is followed by density gradient centrifugation to collect the immune cells for further analysis.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. Prepare density centrifugation media mix solution by placing 90 ml of 10x PBS into 264 ml of ultrapure deionized water (3.93x) in a sterile plastic container. Titrate to pH 7.0-7.2 with HCl and filter sterilize through a 0.22 µm filter. Store at 4 °C. Prepare 70% density centrifugation media by combining 18 ml of density centrifugation medi…

Representative Results

Figure 1: Isolation of Glioma-infiltrating PBMCs. (A) Strategy for the dissection of mouse brain tissue containing early-stage orthotopic glioma implants. The top panel demonstrates the sagittal bisection of the ipsilateral hemisphere away from the contralateral hemisphere. The bottom panel demonstrates the two additional coronal cuts necessary to isolate the target tissue contai…

Materials

Dulbecco's Modification of Eagles Medium	Gibco	12430-054	With 4.5g/L D-glucose, L-glutamine, 25mM HEPES and phenol red
Dulbecco's Phosphate-buffered Saline	Gibco	14190-144	Without calcium chloride or Magnesium chloride
Fetal bovine serum	Omega Scientific Inc.	FB-11
Penicillin Streptomycin	Gibco	15140-122	10,000U/ml Penicillin; 10,000μg/ml Streptomycin
0.6ml conical polypropylenemi microtubes	Genesee Scientific	22-272
Polyurethane ice bucket	Fisherbrand	02-591-45	Capacity: 0.152 oz. (4.5L)
Collagenase (Type I-S)	Sigma Aldrich	C1639	From Clostridium histolyticum
Deoxyribonuclease I	Worthington Biochemical Corp.	LS002007	>2,000 Kunitz units per mg dry weight
Large dissection scissors	Ted Pella Inc.	1316
Small dissection scissors	FST	14094-11
Blunt end forceps	Ted Pella Inc.	13250
7ml glass Dounce tissue grinder	Kontes	KT885300-0007
Percoll Density Centrifugation Media	GE Healthcare	GE17-0891-01
10 mL serological pipettes	Genesee Scientific	12-104	Single use; sterile
70μm sterile nylon mesh cell strainers	Fisherbrand	22363548
15ml polypropylene centrifuge tubes	Genesee Scientific	21-103	Conical bottom
50ml polypropylene centrifuge tubes	Genesee Scientific	21-108	Conical bottom
12x75mm round-bottom polypropylene FACS tubes	Fisherbrand	14-956-1B