Comment[ArrayExpressAccession]	E-MTAB-459
Investigation Title	Cell surface markers that distinguish human beta cells and alpha cells
Comment[Submitted Name]	Cell surface markers that distinguish human beta cells and alpha cells
Experiment Description	The goal of this experiment is to determine cell surface markers that are present in human beta cells or alpha cells but not both. Cells from human islets were FACS sorted and  prepared as matched pairs of beta and alpha cells preparations.
Experimental Design	cell_type_comparison_design	in_vitro_design
Comment[AEExperimentType]	transcription profiling by array
Experimental Factor Type	cell_type
Experimental Factor Name	CELL_TYPE
Person Last Name	Grompe	Dorrell	Schug
Person First Name	Markus	Craig	Jonathan
Person Email	grompem@ohsu.edu	dorrellc@ohsu.edu	jschug@mail.med.upenn.edu
Person Phone	503-494-6888	503-494-6889	215-898-0773
Person Address	L321, 3181 SW Sam Jackson Park Rd. Portland, OR 97239-3098	L321, 3181 SW Sam Jackson Park Rd. Portland, OR 97239-3098	752A CRB, 415 Curie Blvd, Philadelphia, PA 19104-6145
Person Affiliation	Oregon Health Sciences University Oregon Stem Cell Center	Oregon Health & Science University Molecular and Medical Genetics	University of Pennsylvania Functional Genomics Core
Person Roles	submitter	investigator	investigator
Public Release Date	2011-06-10
Protocol Name	P-MTAB-18362	P-MTAB-18363	P-MTAB-18364	P-MTAB-18365	P-MTAB-18366	P-MTAB-18367	P-MTAB-18368	P-MTAB-18369	P-MTAB-18370	P-MTAB-18371	P-MTAB-18372
Protocol Type	purify	fractionate	nucleic_acid_extraction	labeling	pool	hybridization	image_acquisition	feature_extraction	flag_filter	loess_global_normalization	dye_swap_merge
Protocol Description	Islets were collected after 100-700 min of cold ischemia and cultured in CRML for 6-48h prior to over-night shipment. Cell viability on arrival (assessed by Trypan blue staining) was 95-99%. For preparation of a single cell suspension, islets were washed with DPBS (Hyclone, Logan, UT) before incubation in 3 ml 0.05% HyQ Trypsin (Hyclone). Digestion was continued for 10 min. at 37C with gentle dispersal by pipetting with a p1000 micropipettor every 3 min. Undispersed material was removed by passage through a 40 µm strainer (BD Falcon, Bedford, MA) and 1 ml fetal bovine serum (FBS) was slowly added to wash cells  through the strainer, inhibit trypsin activity, and gradually increase the calcium concentration. Cells were then washed and resuspended in serum-free CMRL for brief storage prior to antibody labeling or mouse immunization.	Dissociated cells from primary islets were resuspended in 100 ul DPBS + 2% FBS, combined with an equal volume of hybridoma supernatant and stored at 4C for 30 minutes. Cells were then washed with cold DPBS and resuspended in 100 ul DPBS + 2% FBS containing propidium iodide and a 1:200 dilution of secondary antibody. Secondary antibodies employed included anti-mouse IgG (H+L) conjugated to APC or PE, PE-conjugated anti-mouse IgM (u chain-specific) and APC-conjugated anti-mouse IgG subclasses 1+2a+3 (Jackson ImmunoResearch, West Grove, PA). Dead cells were stained with Propidium iodide (10 ug/ml). For combined intra-/extracellular labeling, cells were then fixed for 20 minutes in 2% paraformaldehyde, washed in DPBS, incubated with antibodies recognizing human amylase or insulin (Santa Cruz) or human c-peptide (BCBC), followed by a suitable secondary antibody (anti-rabbit FITC or anti-rat FITC, Jackson ImmunoResearch). Post-fixation washes and incubations were performed in the presence of 0.5% saponin (Sigma-Aldrich) and 2% FBS. Cells were analyzed using a Becton Dickinson FACScalibur or sorted with a Cytopeia inFluxV-GS, and where possible electronic gating was used to exclude cell doublets (by FSC : Pulse width ratio) from analysis or collection.	Extracted cells or dissected tissue were homogenized in Trizol.  Following chlorophorm extraction, 1 volume 70% EtOH was added to the aqueous phase and applied to RNeasy column (Qiagen). Extraction followed manufacturer recommendations.	Total RNA and 2.5 ug oligo dT are brought to 25 ul and denatured for 5 min at 70 C. The reaction is then cooled to 42 C and an equal volume of RT reaction mix, 400 U SuperScript II,  modified nucleotides, and 20 U RNasin is added. After appropriate incubation, the reaction is denatured, then neutralized, the buffer removed, and the cDNA dried. Dyes are coupled to the cDNA in the dark at room temperature (1 h). The reaction is then quenched.	Biomaterials were pooled.	Hybridization mixture (25 uL) was prepared by combining labeled cDNA (7 uL) with 3 uL human Cot-1 DNA (1 ug/uL; Invitrogen), 2.5 uL deposition control targets (Agilent), and 12.5 uL 2X hybridization buffer (Agilent). The hybridization solution was incubated for 2 min at 98 C to denature the cDNA probe and then cooled to room temperature. The hybridization mixture was centrifuged for 5 minutes at 13,000g, applied to an Agilent Human 1 cDNA microarray, and incubated for 17 h at 65 C. After incubation, arrays were submerged briefly in 0.5X SSC, 0.01% SDS to remove the coverslip, and then washed once in 0.5X SSC, 0.01% SDS for 6 min with agitation and once in 0.06X SSC for 3 min with agitation. Arrays were dried by centrifugation at room temperature for 2 min at 400g.	Image acquisition was performed with the DNA Microarray Scanner Model #G2565BA	Agilent Feature Extraction Software allows extraction of feature intensities from Agilent and non-Agilent 1x3 glass slide microarrays with options to background correct, flag outliers, and normalize.	Filter saturated, non-uniform, and manually flagged spots (Agilent Feature Extraction Software)	The limma's normalize within arrays with method=loess	IDye-swap merging of M (=log(Ch1)-log(Ch2)) values takes as input, for each spot, its values say M and M', from a pair of dye-swap 2-channel assays and outputs (M-M')/2.
Protocol Parameters		cell_type						Software Version		foreground_measurement; numerator_channel; R version; limma version; weights; denominator_channel; background_measurement	numerator; denominator
SDRF File	E-MTAB-459.sdrf.txt
PubMed ID	21632826
Publication Author List	Dorrell, Craig;
Erker, Laura; Schug, Jonathan; Kopp, Janel L.; Canaday, Pamela S.; Fox, Alan J.; Smirnova,
Olga; Duncan, Andrew W.; Finegold, Milton J.; Sander, Maike; Kaestner, Klaus H.; Grompe,
Markus
Publication Title	Prospective isolation of a bipotential clonogenic liver
progenitor cell in adult mice