dispase  (Worthington Biochemical)

Journal: Nature

Article Title: Single Cell Transcriptomics Reconstructs Fate Conversion from Fibroblast to Cardiomyocyte

doi: 10.1038/nature24454

Figure Lengend Snippet: Heterogeneity of our isolated CFs (Thy1+ non-immune non-myocyte cardiac cells) and stepwise suppression of non-cardiomyocyte lineages during iCM reprogramming Related to . ( a-c ) Limited transcriptome change by retrovirus transduction. To determine whether introduction of viruses could influence cellular identities of CF, molecular features of the uninfected and DsRed-transduced cells were compared and only 25 genes were differentially expressed (ANOVA p value

Article Snippet: Neonatal CMs were isolated using the neonatal cardiomyocytes isolation system (Worthington Biochemical Corporation) except that all enzymes were used at a ¼ of the recommended concentration to increase cell viability.

Techniques: Isolation, Transduction

Journal: Nature

Article Title: Single Cell Transcriptomics Reconstructs Fate Conversion from Fibroblast to Cardiomyocyte

doi: 10.1038/nature24454

Figure Lengend Snippet: Heterogeneity of CF and stepwise suppression of non-cardiomyocyte lineages during iCM induction ( a-b ) HC (a) and PCA (b) of control CFs with representative gene expression and GO analysis of the five identified gene clusters. ( c ) HC calculated with control CFs (a) applied to M+G+T-transduced cells with representative gene expression. ( d-f ) 40× ICC images (d,e) with quantifications (f) of Thy1 and SM22α co-stained with αMHC-GFP during reprogramming. n=20 images, scale bar=100 μm, error bars indicate SEM.

Article Snippet: Neonatal CMs were isolated using the neonatal cardiomyocytes isolation system (Worthington Biochemical Corporation) except that all enzymes were used at a ¼ of the recommended concentration to increase cell viability.

Techniques: Expressing, Immunocytochemistry, Staining

Journal: Journal of Synchrotron Radiation

Article Title: High-speed raster-scanning synchrotron serial microcrystallography with a high-precision piezo-scanner

doi: 10.1107/S1600577518010354

Figure Lengend Snippet: Crystallography data collection and processing at FMX. Raster scans of ( a ) a bovine trypsin rod-shaped crystal and ( b ) 5–10 µm-sized proteinase K crystals loaded on MiTeGen loops. The raster-scan areas are shown as red-dashed rectangles; the corresponding heat maps acquired after the raster scans are shown in the insets. The grid coloring is the diffraction pattern spot count. ( c ) Clustering of the serial crystallography partial datasets based on unit-cell size. The data shown were acquired from raster scans of proteinase K microcrystals with a 200 Hz detector frame rate. Of these 279 partial datasets, 234 (blue dots) were indexed to c = 106.58 ± 0.41 Å, with 99.6% completeness (Table 1 ▸ ). The remaining 45 partial datasets (orange dots) with c = 110.06 ± 0.84 Å did not yield a complete dataset and were excluded from refinement. The unit-cell size was converted to Cartesian coordinates for better visualization. The data were clustered into two groups using a K -mean clustering algorithm. ( d ) Hierarchical cluster-analysis dendrogram for the proteinase K partial datasets acquired at a 750 Hz detector frame rate. The cutoff limit was set to 0.6 (dashed line).

Article Snippet: Proteinase K crystals were grown on siliconized glass cover slips (hanging drop) by equilibrating 25 mg ml−1 proteinase K (Worthington Biochemical; LS004224) in 40 mM calcium chloride, 400 mM sodium nitrate and 50 mM BisTris (pH 6.5) over a reservoir containing 160 mM calcium chloride, 1.6 M sodium nitrate and 200 mM BisTris (pH 6.5).

Techniques:

Journal: Nucleic Acids Research

Article Title: Identification of a region in the coiled-coil domain of Smc3 that is essential for cohesin activity

doi: 10.1093/nar/gkw539

Figure Lengend Snippet: Proteolytic cleavage of Smc3 wild-type and mutant proteins. Smc3-V5 and smc3-L217P-3V5 were pulled down by using antibodies against V5. The precipitated proteins were washed and incubated with V8 protease at 23°C in the absence ( A ) or presence ( B ) of 1 mM ATP. The reactions were stopped at the indicated time points. The cleaved products were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and analyzed by western blot, using antibody against V5. The major cleaved products discussed in the text are labeled as a–e. * indicates a non-specific band. A representative experiment is shown. ( n = 4).

Article Snippet: Smc3-3V5 or smc3-L217P-3V5 were precipitated from asynchronous cell cultures and incubated with V8 protease, with or without ATP.

Techniques: Mutagenesis, Incubation, Polyacrylamide Gel Electrophoresis, Western Blot, Labeling

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Primary Culture of Mammalian Taste Epithelium

doi: 10.1007/978-1-62703-125-7_7

Figure Lengend Snippet: Attachment and morphology of cultured human fungiform taste cells. ( a ) Primary cell cultures grown on collagen type-1-coated plates were imaged after 2 days. ( b ) Cells from human fungiform papillae grew for up to 2–4 weeks under attached cell clusters. ( c and d ) Represent day 2 and 4 weeks after harvesting, respectively. During this period we did not observe growth of cells with the appearance of non-taste epithelial cells.

Article Snippet: Enzyme mixture: Mix collagenase type 1 (550 U/mL, Worthington), elastase (10 U/mL, Worthington), and soy bean trypsin inhibitor (0.9 mg/mL, Worthington) in calcium-free Ringer solution just before use.

Techniques: Cell Culture