The purification procedures are based on previously described dissociation (Huettner and Baughman, 1986; Segal et al., 1998) and immunopanning purification protocols for other cell types (Barres et al., 1988; Barres et al., 1992; Meyer-Franke et al., 1995). All aspects of the panning and FACS purification procedures are summarized below.; Preparation Of Mouse Forebrain Cell Suspensions: Six to eight mice from a wild-type litter (C57BL/6, Charles River, Wilmington, MA) or S100β-EGFP transgenic litter (C57BL/6 x DBA (F1), Kosmos line, Zuo et al., 2004) were used. The forebrain was isolated by removal of the olfactory lobes, cerebellum, and midbrain/hindbrain structures by crude dissection, and the tissue was diced with a curved-blade surgical scalpel (Feather, Osaka, Japan, GRF-2976 #10). To isolate cerebral cortical gray matter astrocytes, the brain was sliced in 2-3 mm coronal sections and the cerebral cortex was carefully dissected away from the ventral white matter tracks. This tissue was enzymatically dissociated to make a suspension of single cells, essentially as described by Huettner and Baughman (Huettner and Baughman, 1986; Segal et al., 1998). Briefly, the tissue was incubated at 33 °C for 80 minutes (90 minutes for animals P16 and older) in 20 ml of a papain solution (20 U/ml, Worthington, Lakewood, NJ, LS03126) prepared in dissociation buffer with EDTA (0.5 mM), and L-cysteine-HCl (necessary to activate the papain, 1 mM, Sigma, St. Louis, MO, C7880). The dissociation buffer contained Earle’s balanced salts (EBSS, Sigma, E7510), D(+)-glucose (22.5 mM), NaHCO3 (26 mM), and DNase (125U/ml, Worthington, LS002007) and requires careful equilibration with 5% CO2 and 95% O2 gas before use and during papain treatment. When dissociation buffer is exposed to room air during trituration, minimizing surface area and avoiding bubbles is essential to maintain the proper pH and cell health.; After papain treatment to loosen contacts in the extracellular matrix, the tissue was washed with 3 x 4 ml dissociation buffer containing BSA (1.0 mg/ml, Sigma, A-8806) and ovomucoid (also known as Trypsin Inhibitor, 1.0 mg/ml, Roche Diagnostics Corporation, Indianapolis, IN, 109878) (inhibitor solution) and then mechanically dissociated by gentle sequential trituration using a 5 ml pipette with 5 x 4 ml fresh inhibitor solution to yield a suspension of single cells. Dissociated cells were layered on top of 12 ml of concentrated inhibitor solution (5 mg/ml BSA and 5 mg/ml ovomucoid) and harvested by centrifugation (140 x g for 5 minutes, 220 x g for 10 minutes when purifying OL lineage cells). This method routinely yielded ~15-20 million cells per mouse pup forebrain, with excellent cell health as determined by morphology and viability (>90% by trypan blue exclusion).
Growth protocol
Preparation Of Mouse Astroglia: Preparation of astroglia cultures was as follows. Cortices were isolated by crude dissection of six P1 mice from a S100β-EGFP transgenic litter, meninges were removed, and the tissue was chopped using a curved-blade surgical scalpel. This tissue was enzymatically dissociated to make a suspension of single cells. Briefly, the tissue was incubated at 33 °C for 75 minutes in 20 ml of a papain solution prepared in dissociation buffer containing EDTA and L-cysteine.; After papain treatment the tissue was washed with 3 x 4 ml of DPBS containing BSA (1.5 mg/ml), ovomucoid (1.5 mg/ml), and DNase (125U/ml) (ovomucoid inhibitor). The tissue was then mechanically dissociated by gentle sequential trituration using a 5 ml pipette with 5 x 4 ml fresh ovomucoid inhibitor to yield a suspension of single cells. Dissociated cells were harvested by centrifugation at 220 x g for 10 minutes. Cells were resuspended in 12 ml of concentrated ovomucoid inhibitor (5 mg/ml BSA and 5 mg/ml ovomucoid) and harvested by centrifugation. Dissociated cells were resuspended in 15 ml of astroglia media and plated in 75 cm2 tissue culture flasks (BD Falcon, 353136) that were pre-coated with PDL (10 μg/ml in water, Sigma, P6407) at a density of 25-30 million cells per flask. Astroglia media contained Dulbecco's modified eagle medium (DMEM, Invitrogen, 11960-044), 10% fetal bovine serum (FBS, Invitrogen, 10437-028), 2 mM glutamine (Invitrogen, 25030-081), 1 mM Na pyruvate (Invitrogen, 11360-070), 5 μg/ml N-Acetyl-L-cysteine (NAC, Sigma, A8199), 5 μg/ml insulin (Sigma, I6634), 100 U/ml penicillin-streptomycin (Invitrogen, 15140-122), and 10 μM hydrocortisone (Sigma, H0888). When plated under these conditions most cells including neurons and mature astrocytes die, but a small percentage of immature cells are able to survive and proliferate.; After 3 days the astroglia layer was confluent and the flask was rinsed once with DPBS and then shaken 3 times with DPBS containing phenol red to remove contaminating OL progenitor cells (OPCs), and fresh astroglia media was added. OPCs grow on top of the single cell monolayer of astroglia, so they can be removed by vigorous shaking of the sealed tissue culture flask. After 2 days, 10 μM cytosine arabinoside (AraC, Sigma, C1768) was added to kill dividing cells such as fibroblasts and microglia; the underlying monolayer of astrocytes is not affected by the antimitotic because astroglia divide slowly in dense cultures due to contact inhibition. After 48 hours the astroglia were trypsinized off and plated onto PDL-coated 15 cm tissue culture dishes at 2 million cells per dish in astroglia media.; For preparation of astroglia grown in serum free media, confluent cultures of astroglia prepared as described above were washed three times with DPBS and 20 ml serum-free media was added. Serum-free medium, modified from Bottenstein and Sato (1979), contained neurobasal (Invitrogen, 21103-049), bovine serum albumin (100 μg/ml, BSA, Sigma, A4161), selenium (40 ng/ml, Sigma, S5261), putrescine (16 μg/ml, Sigma, P5780), transferrin (100 μg/ml, Sigma, T1147), progesterone (60 ng/ml, Sigma, P8783), triiodo-thyronine (40 ng/ml, Sigma, T6397), penicillin-streptomycin (100U/ml), insulin (5 μg/ml) NAC (5 μg/ml), pyruvate (1 mM), and glutamine (2 mM).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from acutely purified cells with the RNeasy micro kit (Qiagen, Valencia, CA) using Qiashredder columns for cell lysis and Qiagen on-column DNase treatment to remove any contaminating genomic DNA. The integrity of RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara , CA), and RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Rockland, DE).
Label
biotin
Label protocol
Biotinylated cRNAs for hybridization to Affymetrix 3'-arrays were prepared from 30-50 ng total RNA using the Affymetrix two-cycle target labeling assay with spike in controls (Affymetrix Inc., Santa Clara, CA, 900494).
Hybridization protocol
Labeled-cRNA was fragmented and hybridized to Mouse Genome 430 2.0 Arrays (3'-arrays, Affymetrix, 900495) following the manufacturer’s protocols at the Stanford Protein and Nucleic Acid Facility
Scan protocol
Hybridized Mouse Genome 430 2.0 Arrays (3'-arrays, Affymetrix, 900495) were scanned following the manufacturer’s protocols at the Stanford Protein and Nucleic Acid Facility
Description
John_34A-P1-pos_Mouse430 _2
Data processing
Raw image files were processed using Affymetrix GCOS 1.3 software to calculate individual probe cell intensity data and generate CEL data files. Using GCOS and the MAS 5.0 algorithm, intensity data was normalized per chip to a target intensity TGT value of 500, and expression data and present/absent calls for individual probe sets were calculated. Gene symbols and names for data analyzed with the MAS 5.0 algorithm were from the Affymetrix NetAffx Mouse430_2 annotations file (http://www.affymetrix.com/support/technical/byproduct.affx?product=moe430-20). Quality control was performed by examining raw DAT image files for anomalies, confirming each GeneChip array had a background value less than 100, monitoring that the percentage present calls was appropriate for the cell type, and inspecting the poly(A) spike in controls, housekeeping genes, and hybridization controls to confirm labeling and hybridization consistency. In addition, the quantity of RNA purified per cell, the labeling efficiency for each cycle of amplification, and the fragmentation efficiency were carefully monitored.; CEL data files were also independently analyzed using the dChip algorithm. Gene expression values were normalized and modeled across arrays using the dChip software package with invariant-set normalization and a PM model (www.dchip.org, Li and Wong, 2001).
