Journal: Cell stem cell

Article Title: Sliced Human Cortical Organoids for Modeling Distinct Cortical Layer Formation

doi: 10.1016/j.stem.2020.02.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: BrainPhys neuronal medium , StemCell Technologies , cat. # 05790.

Techniques: Recombinant, Saline, Knock-Out, Protease Inhibitor, Imaging, DNA Library Preparation, Plasmid Preparation, Derivative Assay, Mutagenesis, TALENs, Software, RNA Sequencing

Journal: Nature Communications

Article Title: BrainPhys neuronal medium optimized for imaging and optogenetics in vitro

doi: 10.1038/s41467-020-19275-x

Figure Lengend Snippet: a The osmolality of BrainPhys Imaging (BPI) is similar to human cerebrospinal fluid (hCSF) (~305 mOsmol/kg). Neurobasal and NEUMO were significantly lower (210–220 mOsmol/kg) and DMEM/F12 and FluoroBrite higher (317–338 mOsmol/kg). Media osmolality data was collected from four replicates per condition. A total of three hCSF samples, each pooled from up to four subjects, were tested across three independent experiments ( n = 3). See also Supplementary Fig. . b – f Comparison of the optical properties of BPI with other basal media specialized for imaging (NEUMO, FluoroBrite), standard basal neuromedia (BrainPhys, BP; BrainPhys without phenol red, BP no PR; DMEM/F12; Neurobasal) and control media (phosphate-buffered saline, PBS; deionized water, H 2 O). b The absorbance spectra from 300 to 800 nm acquired from basal media alone (without cells), and after adding supplements required for the long-term culture of brain cells. Virtually all fluorophores used for cell imaging require light stimulation above 300 nm. c – f The mean autofluorescence intensities of basal media (without cells) across the entire visible spectrum. BPI shows autofluorescence intensities similar to PBS. c The emission spectra across 400–700 nm captured for the 375 (ultra-violet), 405 (violet), 488 (blue), and 532 nm (green) excitation wavelengths from test and control media. d – f Autofluorescence at 460, 520, and 590 nm emission wavelengths were measured following excitation at 355, 485, and 544 nm, respectively. Results were generated from eight replicate wells per medium ( n = 8) analyzed across three independent experiments. For normalization, the mean fluorescence intensity in PBS was subtracted from the other media. Data are presented as mean ± SEM. Significance determined via two-tailed nonparametric unpaired (Mann–Whitney) tests. ns, P > 0.05.

Article Snippet: BrainPhys™ (Cat. No. 05790, STEMCELL Technologies), BrainPhys™ Imaging (Cat. No. 05796, STEMCELL Technologies), BrainPhys without Phenol Red (Cat. No. 05791, STEMCELL Technologies), FluoroBrite™ DMEM (Cat. No. A1896701, Thermo Fisher Scientific), DMEM/F12 (Cat. No. 10565018, Thermo Fisher Scientific), Neurobasal (Cat. No. 21103-049, Thermo Fisher Scientific), and BrightCell™ NEUMO (Cat. No. SCM146, Merck) were obtained via the source and catalog details provided.

Techniques: Imaging, Generated, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: BrainPhys neuronal medium optimized for imaging and optogenetics in vitro

doi: 10.1038/s41467-020-19275-x

Figure Lengend Snippet: a The osmolality of BrainPhys Imaging (BPI) is similar to human cerebrospinal fluid (hCSF) (~305 mOsmol/kg). Neurobasal and NEUMO were significantly lower (210–220 mOsmol/kg) and DMEM/F12 and FluoroBrite higher (317–338 mOsmol/kg). Media osmolality data was collected from four replicates per condition. A total of three hCSF samples, each pooled from up to four subjects, were tested across three independent experiments ( n = 3). See also Supplementary Fig. . b – f Comparison of the optical properties of BPI with other basal media specialized for imaging (NEUMO, FluoroBrite), standard basal neuromedia (BrainPhys, BP; BrainPhys without phenol red, BP no PR; DMEM/F12; Neurobasal) and control media (phosphate-buffered saline, PBS; deionized water, H 2 O). b The absorbance spectra from 300 to 800 nm acquired from basal media alone (without cells), and after adding supplements required for the long-term culture of brain cells. Virtually all fluorophores used for cell imaging require light stimulation above 300 nm. c – f The mean autofluorescence intensities of basal media (without cells) across the entire visible spectrum. BPI shows autofluorescence intensities similar to PBS. c The emission spectra across 400–700 nm captured for the 375 (ultra-violet), 405 (violet), 488 (blue), and 532 nm (green) excitation wavelengths from test and control media. d – f Autofluorescence at 460, 520, and 590 nm emission wavelengths were measured following excitation at 355, 485, and 544 nm, respectively. Results were generated from eight replicate wells per medium ( n = 8) analyzed across three independent experiments. For normalization, the mean fluorescence intensity in PBS was subtracted from the other media. Data are presented as mean ± SEM. Significance determined via two-tailed nonparametric unpaired (Mann–Whitney) tests. ns, P > 0.05.

Article Snippet: BrainPhys™ (Cat. No. 05790, STEMCELL Technologies), BrainPhys™ Imaging (Cat. No. 05796, STEMCELL Technologies), BrainPhys without Phenol Red (Cat. No. 05791, STEMCELL Technologies), FluoroBrite™ DMEM (Cat. No. A1896701, Thermo Fisher Scientific), DMEM/F12 (Cat. No. 10565018, Thermo Fisher Scientific), Neurobasal (Cat. No. 21103-049, Thermo Fisher Scientific), and BrightCell™ NEUMO (Cat. No. SCM146, Merck) were obtained via the source and catalog details provided.

Techniques: Imaging, Generated, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: BrainPhys neuronal medium optimized for imaging and optogenetics in vitro

doi: 10.1038/s41467-020-19275-x

Figure Lengend Snippet: Intracellular Ca 2+ changes in human PSC-derived neurons were measured with time-lapse image sequences of a Ca 2+ sensor (Fluo-4 AM). A total 1225 human neurons were analyzed across thirteen fields of view (FOVs) from four coverslips ( n = 4 biologically independent experiments). Regions of interest (ROIs) were drawn on the cell soma to determine fluorescence intensity changes (Δ F / F 0 ) over time. Active events (Δ F / F 0 > 5% from baseline) were manually categorized into fast-rising Ca 2+ spikes or slow-rising Ca 2+ waves (see Fig. ). The same FOVs were imaged in artificial cerebrospinal fluid (ACSF), BPI, FluoroBrite, and NEUMO. a Example Fluo-4 AM fluorescence image of a neuronal population in BPI. White circles represent active ROIs. Fluorescent intensity represents intracellular Ca 2+ levels. b Typical fast-rising Ca 2+ spikes from five active cells in BPI. c , d No significant difference was observed between the average properties of fast-rising unitary Ca 2+ spike events in BPI ( n = 16 cells) and ACSF ( n = 24 cells) from six FOVs across two coverslips. e , g No significant difference was observed between the proportions of cells with fast Ca 2+ spikes or slow Ca 2+ waves in BPI and ACSF. The total active cells (BPI: n = 329; ACSF n = 308) were compared across six FOVs from two coverslips. Voltage-gated sodium channel blocker Tetrodotoxin (TTX, 1 µM) significantly reduced Ca 2+ spikes and waves. See also Supplementary Fig. . i , k Comparison of Ca 2+ signals in BPI, NEUMO, and FluoroBrite show significant differences in the proportions of cells with Ca 2+ spikes but not waves. Active cells with Ca 2+ spikes and spikes/waves in BPI ( n = 243 cells), FluoroBrite ( n = 39 cells) and NEUMO ( n = 18 cells) or with Ca 2+ waves in BPI ( n = 288 cells), FluoroBrite ( n = 302 cells), and NEUMO ( n = 356 cells) were compared across seven FOVs from two coverslips. f , h , j , l Example traces from the same ROIs in different media. Symbols in c – e , g , i , k represent cells recorded first (triangles), second (circles), third (square), fourth (diamond) in either medium. Values are presented as mean ± SEM. Significance in c – e , g , i , k determined via two-tailed nonparametric unpaired (Mann–Whitney) tests. ns, P > 0.05.

Article Snippet: BrainPhys™ (Cat. No. 05790, STEMCELL Technologies), BrainPhys™ Imaging (Cat. No. 05796, STEMCELL Technologies), BrainPhys without Phenol Red (Cat. No. 05791, STEMCELL Technologies), FluoroBrite™ DMEM (Cat. No. A1896701, Thermo Fisher Scientific), DMEM/F12 (Cat. No. 10565018, Thermo Fisher Scientific), Neurobasal (Cat. No. 21103-049, Thermo Fisher Scientific), and BrightCell™ NEUMO (Cat. No. SCM146, Merck) were obtained via the source and catalog details provided.

Techniques: Derivative Assay, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: BrainPhys neuronal medium optimized for imaging and optogenetics in vitro

doi: 10.1038/s41467-020-19275-x

Figure Lengend Snippet: a The osmolality of BrainPhys Imaging (BPI) is similar to human cerebrospinal fluid (hCSF) (~305 mOsmol/kg). Neurobasal and NEUMO were significantly lower (210–220 mOsmol/kg) and DMEM/F12 and FluoroBrite higher (317–338 mOsmol/kg). Media osmolality data was collected from four replicates per condition. A total of three hCSF samples, each pooled from up to four subjects, were tested across three independent experiments ( n = 3). See also Supplementary Fig. . b – f Comparison of the optical properties of BPI with other basal media specialized for imaging (NEUMO, FluoroBrite), standard basal neuromedia (BrainPhys, BP; BrainPhys without phenol red, BP no PR; DMEM/F12; Neurobasal) and control media (phosphate-buffered saline, PBS; deionized water, H 2 O). b The absorbance spectra from 300 to 800 nm acquired from basal media alone (without cells), and after adding supplements required for the long-term culture of brain cells. Virtually all fluorophores used for cell imaging require light stimulation above 300 nm. c – f The mean autofluorescence intensities of basal media (without cells) across the entire visible spectrum. BPI shows autofluorescence intensities similar to PBS. c The emission spectra across 400–700 nm captured for the 375 (ultra-violet), 405 (violet), 488 (blue), and 532 nm (green) excitation wavelengths from test and control media. d – f Autofluorescence at 460, 520, and 590 nm emission wavelengths were measured following excitation at 355, 485, and 544 nm, respectively. Results were generated from eight replicate wells per medium ( n = 8) analyzed across three independent experiments. For normalization, the mean fluorescence intensity in PBS was subtracted from the other media. Data are presented as mean ± SEM. Significance determined via two-tailed nonparametric unpaired (Mann–Whitney) tests. ns, P > 0.05.

Article Snippet: BrainPhys™ (Cat. No. 05790, STEMCELL Technologies), BrainPhys™ Imaging (Cat. No. 05796, STEMCELL Technologies), BrainPhys without Phenol Red (Cat. No. 05791, STEMCELL Technologies), FluoroBrite™ DMEM (Cat. No. A1896701, Thermo Fisher Scientific), DMEM/F12 (Cat. No. 10565018, Thermo Fisher Scientific), Neurobasal (Cat. No. 21103-049, Thermo Fisher Scientific), and BrightCell™ NEUMO (Cat. No. SCM146, Merck) were obtained via the source and catalog details provided.

Techniques: Imaging, Generated, Fluorescence, Two Tailed Test, MANN-WHITNEY