Journal: bioRxiv

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

doi: 10.1101/2020.09.02.276535

Figure Lengend Snippet: A, shows that the osmolality of BrainPhys Imaging (BPI) is around 305 mOsmol/kg, which is similar to physiological human cerebrospinal fluid (hCSF). Neurobasal and NEUMO were significantly lower (210-220 mOsmol/kg) and DMEM/F12 and FluoroBrite higher (317-338 mOsmol/kg). Data was collected from 3-4 replicates per condition. B-F, compares the optical properties of BPI with other basal media specialized for imaging (NEUMO, FluoroBrite), standard basal neuro-media (BrainPhys “BP”, BrainPhys without phenol red “BP (w/out PR)”, DMEM/F12, Neurobasal) and control media (phosphate-buffered saline (PBS) and deionized water (H 2 O)). B, shows the absorbance spectra from 300-800 nm acquired from basal media alone (without cells), and after adding supplements required for the long-term culture of brain cells. BPI shows lower absorbance than all other culture media tested. Adding supplements to BPI did not increase the absorbance, which was tested between 300-800 nm. Virtually all fluorophores used for cell imaging require light stimulation above 300 nm. C-F, reveals the mean auto-fluorescence intensities of basal media (without cells) across the entire visible spectrum. BPI shows auto-fluorescence intensities similar to PBS. C, shows the emission spectra across 400-700 nm captured for the 375 (violet), 405 (blue), 488 (green) and 532 nm (red) 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 across three independent experiments. For normalization, the mean fluorescence intensity in PBS was subtracted from the other media. Values represent mean ± SEM. Significance determined via two-tailed non-parametric unpaired (Mann Whitney) tests. P-values are annotated as follows: * for P<0.05, ** for P<0.01, and ns for P>0.05.

Article Snippet: 5 days post-plating, half media changes were performed every 3-4 days with either BrainPhys™ (STEMCELL Technologies), Neurobasal™, BrightCell™ NEUMO (Merck) BrainPhys™ Without Phenol Red (STEMCELL Technologies), or BrainPhys™ Imaging (STEMCELL Technologies) supplemented with 1x SM1.

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

Journal: bioRxiv

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

doi: 10.1101/2020.09.02.276535

Figure Lengend Snippet: Fluorescent imaging in BPI medium improves signal-to-background ratios relative to BrainPhys medium. For signal-to-background ratio quantification, mean intensity ‘signal’ values at nuclei (Hoechst), combined neurites and soma (NeuO, β-III tubulin, GFP), or isolated neurite and soma (GFP) regions of interest (ROI) were measured and compared to mean ‘background’ intensities. For each field-of-view (FOV), signal-to-background ratios were calculated using the following formula: [(signal intensity – background intensity)/background intensity]. A-C, BPI improves signal-to-background ratios at soma and/or neurite regions and reduces background intensities relative to BP. A, representative fluorescent images of live human iPSC-derived neurons expressing GFP (excitation/emission peaks: 488/509 nm; filters: 470±40/525±50 nm) and imaged in BPI, BP or artificial cerebrospinal fluid (ACSF) using a 40x water immersion lens (0.8 NA). Higher mean GFP intensities at soma and neurite regions were observed in BPI and ACSF relative to BP, whereas background intensities were reduced. B-C, the analysis was conducted on GFP images from 9 field-of-views of the same cells in BrainPhys, BPI or ACSF. All data is paired. B, left: shows the cumulative percentage (%) of pixels at background, soma and neurite regions in BP (grey), BPI (blue), and ACSF (black) above a range of mean GFP intensity thresholds. Camera dark counts without medium and cells are labeled as ‘dark’ (top panel). B, right: shows that mean intensity values at background regions (top panel) in ACSF and BPI were significantly reduced relative to BP. Signal-to-background ratios at isolated neurite and soma regions were significantly higher in BPI and ACSF than BP. Combining neurite and soma regions in (C) also showed improved signal-to-background ratios in BPI and ACSF compared to BP. D, representative images of brain assembloids representing a GFP-labelled ventral organoid fused with a non-labeled dorsal organoid imaged using 488nm laser light in Organoid Maturation medium (right) or BPI (left). Note the increased visibility of interneuron migration in the organoids imaged in BPI (bottom). Images in both media are displayed with the following minimum/maximum intensity counts: 0/75. E, shows representative fluorescent images of primary cortical neurons stained and imaged live with Hoechst 33342 (excitation/emission peaks: 350/461 nm; filters: 377±25/447±30 nm), live with NeuroFluor NeuO (470/555 nm; 475±17/536±20 nm), and fixed with β-III tubulin-Dylight594 (594/618 nm; 562±20/624±20 nm). Images taken using a 20x air immersion lens (0.45 NA) in both media are displayed with the following minimum/maximum intensity counts: 0/8500 (Hoechst), 0/3600 (NeuO), 1000/12000 (β-III tubulin). F, signal-to-background ratios were improved when imaging Hoechst in BPI (n=23 FOVs) compared to BP (n=24 FOVs). This was also seen for NeuO images captured in BPI (n=23 FOVs) relative to BP (n=22 FOVs). No significant differences were found when imaging β-III tubulin in either BPI (n=8 FOVs) or BP (n=9 FOVs). Images were collected from two biologically independent experiments (Hoescht/NeuO; β-III tubulin) with one well per condition. See also S1. All values represent mean±SEM. B, C, significance determined via two-tailed non-parametric paired (Wilcoxon) tests and (F) non-parametric unpaired (Mann Whitney) tests. P-values are annotated as follows: * for P<0.05, ** for P<0.01, **** for P<0.0001 and ns for P>0.05.

Article Snippet: 5 days post-plating, half media changes were performed every 3-4 days with either BrainPhys™ (STEMCELL Technologies), Neurobasal™, BrightCell™ NEUMO (Merck) BrainPhys™ Without Phenol Red (STEMCELL Technologies), or BrainPhys™ Imaging (STEMCELL Technologies) supplemented with 1x SM1.

Techniques: Imaging, Isolation, Derivative Assay, Expressing, Labeling, Migration, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Developmental cell

Article Title: Loss of Non-motor Kinesin KIF26A Causes Congenital Brain Malformations via Dysregulated Neuronal Migration and Axonal Growth as well as Apoptosis

doi: 10.1016/j.devcel.2022.09.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: BrainPhys TM Neuronal Medium and SM1 Kit , StemCell Technologies , cat. # 05792.

Techniques: Recombinant, Saline, Plasmid Preparation, Lysis, Extraction, RNAscope, Multiplex Assay, Imaging, In Situ, RNA Sequencing, Derivative Assay, Mutagenesis, Knock-Out, CRISPR, Software

Journal: bioRxiv

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

doi: 10.1101/2020.09.02.276535

Figure Lengend Snippet: A, shows that the osmolality of BrainPhys Imaging (BPI) is around 305 mOsmol/kg, which is similar to physiological human cerebrospinal fluid (hCSF). Neurobasal and NEUMO were significantly lower (210-220 mOsmol/kg) and DMEM/F12 and FluoroBrite higher (317-338 mOsmol/kg). Data was collected from 3-4 replicates per condition. B-F, compares the optical properties of BPI with other basal media specialized for imaging (NEUMO, FluoroBrite), standard basal neuro-media (BrainPhys “BP”, BrainPhys without phenol red “BP (w/out PR)”, DMEM/F12, Neurobasal) and control media (phosphate-buffered saline (PBS) and deionized water (H 2 O)). B, shows the absorbance spectra from 300-800 nm acquired from basal media alone (without cells), and after adding supplements required for the long-term culture of brain cells. BPI shows lower absorbance than all other culture media tested. Adding supplements to BPI did not increase the absorbance, which was tested between 300-800 nm. Virtually all fluorophores used for cell imaging require light stimulation above 300 nm. C-F, reveals the mean auto-fluorescence intensities of basal media (without cells) across the entire visible spectrum. BPI shows auto-fluorescence intensities similar to PBS. C, shows the emission spectra across 400-700 nm captured for the 375 (violet), 405 (blue), 488 (green) and 532 nm (red) 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 across three independent experiments. For normalization, the mean fluorescence intensity in PBS was subtracted from the other media. Values represent mean ± SEM. Significance determined via two-tailed non-parametric unpaired (Mann Whitney) tests. P-values are annotated as follows: * for P<0.05, ** for P<0.01, and ns for P>0.05.

Article Snippet: 5 days post-plating, half media changes were performed every 3-4 days with either BrainPhys™ (STEMCELL Technologies), Neurobasal™, BrightCell™ NEUMO (Merck) BrainPhys™ Without Phenol Red (STEMCELL Technologies), or BrainPhys™ Imaging (STEMCELL Technologies) supplemented with 1x SM1.

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

Journal: bioRxiv

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

doi: 10.1101/2020.09.02.276535

Figure Lengend Snippet: Fluorescent imaging in BPI medium improves signal-to-background ratios relative to BrainPhys medium. For signal-to-background ratio quantification, mean intensity ‘signal’ values at nuclei (Hoechst), combined neurites and soma (NeuO, β-III tubulin, GFP), or isolated neurite and soma (GFP) regions of interest (ROI) were measured and compared to mean ‘background’ intensities. For each field-of-view (FOV), signal-to-background ratios were calculated using the following formula: [(signal intensity – background intensity)/background intensity]. A-C, BPI improves signal-to-background ratios at soma and/or neurite regions and reduces background intensities relative to BP. A, representative fluorescent images of live human iPSC-derived neurons expressing GFP (excitation/emission peaks: 488/509 nm; filters: 470±40/525±50 nm) and imaged in BPI, BP or artificial cerebrospinal fluid (ACSF) using a 40x water immersion lens (0.8 NA). Higher mean GFP intensities at soma and neurite regions were observed in BPI and ACSF relative to BP, whereas background intensities were reduced. B-C, the analysis was conducted on GFP images from 9 field-of-views of the same cells in BrainPhys, BPI or ACSF. All data is paired. B, left: shows the cumulative percentage (%) of pixels at background, soma and neurite regions in BP (grey), BPI (blue), and ACSF (black) above a range of mean GFP intensity thresholds. Camera dark counts without medium and cells are labeled as ‘dark’ (top panel). B, right: shows that mean intensity values at background regions (top panel) in ACSF and BPI were significantly reduced relative to BP. Signal-to-background ratios at isolated neurite and soma regions were significantly higher in BPI and ACSF than BP. Combining neurite and soma regions in (C) also showed improved signal-to-background ratios in BPI and ACSF compared to BP. D, representative images of brain assembloids representing a GFP-labelled ventral organoid fused with a non-labeled dorsal organoid imaged using 488nm laser light in Organoid Maturation medium (right) or BPI (left). Note the increased visibility of interneuron migration in the organoids imaged in BPI (bottom). Images in both media are displayed with the following minimum/maximum intensity counts: 0/75. E, shows representative fluorescent images of primary cortical neurons stained and imaged live with Hoechst 33342 (excitation/emission peaks: 350/461 nm; filters: 377±25/447±30 nm), live with NeuroFluor NeuO (470/555 nm; 475±17/536±20 nm), and fixed with β-III tubulin-Dylight594 (594/618 nm; 562±20/624±20 nm). Images taken using a 20x air immersion lens (0.45 NA) in both media are displayed with the following minimum/maximum intensity counts: 0/8500 (Hoechst), 0/3600 (NeuO), 1000/12000 (β-III tubulin). F, signal-to-background ratios were improved when imaging Hoechst in BPI (n=23 FOVs) compared to BP (n=24 FOVs). This was also seen for NeuO images captured in BPI (n=23 FOVs) relative to BP (n=22 FOVs). No significant differences were found when imaging β-III tubulin in either BPI (n=8 FOVs) or BP (n=9 FOVs). Images were collected from two biologically independent experiments (Hoescht/NeuO; β-III tubulin) with one well per condition. See also S1. All values represent mean±SEM. B, C, significance determined via two-tailed non-parametric paired (Wilcoxon) tests and (F) non-parametric unpaired (Mann Whitney) tests. P-values are annotated as follows: * for P<0.05, ** for P<0.01, **** for P<0.0001 and ns for P>0.05.

Article Snippet: 5 days post-plating, half media changes were performed every 3-4 days with either BrainPhys™ (STEMCELL Technologies), Neurobasal™, BrightCell™ NEUMO (Merck) BrainPhys™ Without Phenol Red (STEMCELL Technologies), or BrainPhys™ Imaging (STEMCELL Technologies) supplemented with 1x SM1.

Techniques: Imaging, Isolation, Derivative Assay, Expressing, Labeling, Migration, Staining, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

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

doi: 10.1101/2020.09.02.276535

Figure Lengend Snippet: A, shows that the osmolality of BrainPhys Imaging (BPI) is around 305 mOsmol/kg, which is similar to physiological human cerebrospinal fluid (hCSF). Neurobasal and NEUMO were significantly lower (210-220 mOsmol/kg) and DMEM/F12 and FluoroBrite higher (317-338 mOsmol/kg). Data was collected from 3-4 replicates per condition. B-F, compares the optical properties of BPI with other basal media specialized for imaging (NEUMO, FluoroBrite), standard basal neuro-media (BrainPhys “BP”, BrainPhys without phenol red “BP (w/out PR)”, DMEM/F12, Neurobasal) and control media (phosphate-buffered saline (PBS) and deionized water (H 2 O)). B, shows the absorbance spectra from 300-800 nm acquired from basal media alone (without cells), and after adding supplements required for the long-term culture of brain cells. BPI shows lower absorbance than all other culture media tested. Adding supplements to BPI did not increase the absorbance, which was tested between 300-800 nm. Virtually all fluorophores used for cell imaging require light stimulation above 300 nm. C-F, reveals the mean auto-fluorescence intensities of basal media (without cells) across the entire visible spectrum. BPI shows auto-fluorescence intensities similar to PBS. C, shows the emission spectra across 400-700 nm captured for the 375 (violet), 405 (blue), 488 (green) and 532 nm (red) 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 across three independent experiments. For normalization, the mean fluorescence intensity in PBS was subtracted from the other media. Values represent mean ± SEM. Significance determined via two-tailed non-parametric unpaired (Mann Whitney) tests. P-values are annotated as follows: * for P<0.05, ** for P<0.01, and ns for P>0.05.

Article Snippet: 5 days post-plating, half media changes were performed every 3-4 days with either BrainPhys™ (STEMCELL Technologies), Neurobasal™, BrightCell™ NEUMO (Merck) BrainPhys™ Without Phenol Red (STEMCELL Technologies), or BrainPhys™ Imaging (STEMCELL Technologies) supplemented with 1x SM1.

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

Journal: bioRxiv

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

doi: 10.1101/2020.09.02.276535

Figure Lengend Snippet: Fluorescent imaging in BPI medium improves signal-to-background ratios relative to BrainPhys medium. For signal-to-background ratio quantification, mean intensity ‘signal’ values at nuclei (Hoechst), combined neurites and soma (NeuO, β-III tubulin, GFP), or isolated neurite and soma (GFP) regions of interest (ROI) were measured and compared to mean ‘background’ intensities. For each field-of-view (FOV), signal-to-background ratios were calculated using the following formula: [(signal intensity – background intensity)/background intensity]. A-C, BPI improves signal-to-background ratios at soma and/or neurite regions and reduces background intensities relative to BP. A, representative fluorescent images of live human iPSC-derived neurons expressing GFP (excitation/emission peaks: 488/509 nm; filters: 470±40/525±50 nm) and imaged in BPI, BP or artificial cerebrospinal fluid (ACSF) using a 40x water immersion lens (0.8 NA). Higher mean GFP intensities at soma and neurite regions were observed in BPI and ACSF relative to BP, whereas background intensities were reduced. B-C, the analysis was conducted on GFP images from 9 field-of-views of the same cells in BrainPhys, BPI or ACSF. All data is paired. B, left: shows the cumulative percentage (%) of pixels at background, soma and neurite regions in BP (grey), BPI (blue), and ACSF (black) above a range of mean GFP intensity thresholds. Camera dark counts without medium and cells are labeled as ‘dark’ (top panel). B, right: shows that mean intensity values at background regions (top panel) in ACSF and BPI were significantly reduced relative to BP. Signal-to-background ratios at isolated neurite and soma regions were significantly higher in BPI and ACSF than BP. Combining neurite and soma regions in (C) also showed improved signal-to-background ratios in BPI and ACSF compared to BP. D, representative images of brain assembloids representing a GFP-labelled ventral organoid fused with a non-labeled dorsal organoid imaged using 488nm laser light in Organoid Maturation medium (right) or BPI (left). Note the increased visibility of interneuron migration in the organoids imaged in BPI (bottom). Images in both media are displayed with the following minimum/maximum intensity counts: 0/75. E, shows representative fluorescent images of primary cortical neurons stained and imaged live with Hoechst 33342 (excitation/emission peaks: 350/461 nm; filters: 377±25/447±30 nm), live with NeuroFluor NeuO (470/555 nm; 475±17/536±20 nm), and fixed with β-III tubulin-Dylight594 (594/618 nm; 562±20/624±20 nm). Images taken using a 20x air immersion lens (0.45 NA) in both media are displayed with the following minimum/maximum intensity counts: 0/8500 (Hoechst), 0/3600 (NeuO), 1000/12000 (β-III tubulin). F, signal-to-background ratios were improved when imaging Hoechst in BPI (n=23 FOVs) compared to BP (n=24 FOVs). This was also seen for NeuO images captured in BPI (n=23 FOVs) relative to BP (n=22 FOVs). No significant differences were found when imaging β-III tubulin in either BPI (n=8 FOVs) or BP (n=9 FOVs). Images were collected from two biologically independent experiments (Hoescht/NeuO; β-III tubulin) with one well per condition. See also S1. All values represent mean±SEM. B, C, significance determined via two-tailed non-parametric paired (Wilcoxon) tests and (F) non-parametric unpaired (Mann Whitney) tests. P-values are annotated as follows: * for P<0.05, ** for P<0.01, **** for P<0.0001 and ns for P>0.05.

Article Snippet: 5 days post-plating, half media changes were performed every 3-4 days with either BrainPhys™ (STEMCELL Technologies), Neurobasal™, BrightCell™ NEUMO (Merck) BrainPhys™ Without Phenol Red (STEMCELL Technologies), or BrainPhys™ Imaging (STEMCELL Technologies) supplemented with 1x SM1.

Techniques: Imaging, Isolation, Derivative Assay, Expressing, Labeling, Migration, Staining, Two Tailed Test, MANN-WHITNEY