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M.T. markers that would allow specific studies of the heterogeneous population of neural stem and progenitor cells (collectively labeled NPCs). Existing markers such as nestin, Sox-2, brain lipid-binding protein (BLBP), glial fibrillary acidic protein (GFAP) and others, are expressed not only by NPCs but also by other cell types, mainly of the astroglial lineage4C9. While several other markers appear to be selective for NSCs10C13, they are all intracellular, limiting characterization of these cells to ex-vivo studies. To identify novel markers of these cells and capture possible membrane-bound antigens, we generated several mouse antibodies (Abs) against cultured mouse embryonic neurospheres that contain both neural stem and progenitor cells. The NSC-6 Ab, which produced the most robust staining of NPCs, corresponded to Brain-Abundant, membrane-attached Signal Protein 1 (BASP1), a protein not previously described in NPCs. BASP1 (also known as and A 2-DE gel obtained with 90?g of human hippocampus lysate and stained for total proteins with Sypro Ruby fluorescent stain. Immunoblot obtained from the same protein load run on an identical gel in parallel with that of (B), and immunolabeled with the NSC-6 Ab. The spot of interest, corresponding to molecular weight of 45?kDa, is indicated in the center of the blot. The location of a blank, control spot is also indicated. Separate blots, shown in their entirety, were cropped and separated by white space. (G) ALRH LC/MS/MS analysis indicates that the spot of interest is usually accession # IPI00299024 BASP1, brain abundant, membrane attached signal protein 1. Four peptides were identified in six spectra, with 34.9% coverage. aEach peptide sequence was decided in distinct MS/MS spectra. All six spectra were manually confirmed. bp-values?D-69491 from range, followed by five tandem-mass (MS/MS) events sequentially generated in a data-dependent manner on the first, second, third, fourth, and fifth most intense ions selected from the full MS spectrum (at 35% collision energy). We used the Xcalibur data system (THERMO-FINNIGAN) to control the mass spectrometer scan functions and HPLC solvent gradients. MS/MS spectra were extracted from the RAW file with Readw.exe (http://sourceforge.net/projects/sashimi). The resulting mzXML file contains all the data for all those MS/MS spectra and can be read by the subsequent analysis software. The MS/MS data was searched with Inspect57 against a human IPI database with optional modifications:?+?16 on Methionine,?+?57 on Cysteine,?+?80 on Threonine, Serine and Tyrosine. D-69491 Only peptides D-69491 with at least a p-value of 0.025 were analyzed further. Common contaminants (e.g., keratins) were removed from the returned data set. Proteins identified by at least three distinct peptides within a sample were considered valid; when sample signal was very weak, two distinct peptides were accepted for a valid identification. Further validation of sequences of interest was obtained by manual inspection of the MS/MS spectra. Flow cytometry Plated human iPSC-derived NPCs (hNPCs) were harvested using Accutase cell dissociation solution (GIBCO), washed in ice-cold flow buffer (DPBS, 2% fetal bovine serum, 2?mM EDTA). In each condition, 1??106 cells were incubated with the NSC-6 antibody for 45?min on ice (1:20; optimal concentration determined by previous titration experiments). The cells were then washed twice in ice-cold flow buffer with 2% normal donkey serum added, then stained in the same buffer with Alexa Fluor 405 (1:200 donkey anti-mouse IgG H&L; ABCAM 175659) secondary antibody for 45?min on ice. Stained cells were washed twice in ice-cold flow buffer then resuspended in a viability dye solution (20?mM Helix NP NIR, BIOLEGEND, 425301 in flow buffer) and immediately analyzed D-69491 by flow cytometry using an D-69491 LSR Fortessa (BECTON DICKINSON) with acquisition of AF405 on V450 and Helix-NIR on R670. Final data analyses were performed using FlowJo software (TREE STAR INC.). To determine hNPC NSC-6 positivity, 250,000 live cell events were acquired. NSC-6 positive signal was set by gating for single viable cells and.