mscarlet proteins  (ATCC)

Journal: Lab on a Chip

Article Title: Controlling spatial structure in minimal microbial communities by sequential capillary assembly

doi: 10.1039/d6lc00040a

Figure Lengend Snippet: Nanobody-functionalised silica particles bind selectively to target bacteria. i) Graphical illustration of particle–bacteria binding strategy. The cysteine group on the nanobody is bound to a PEG11 linker via a maleimide bond which, in turn, is bound to streptavidin-functionalised 2.7 μm silica particles via a biotin bond. ii) Particle–bacteria binding after mixing for 30 min in a 1.5 ml Eppendorf and imaging in a 96-well plate in the phase contrast channel or iii) fluorescence channel. White arrow highlights a silica particle, blue arrow highlights a bacterium. a) Sybody-F1 (SbF1) functionalised particles bound to S. aureus GFP. b) Nanobody 01 (Nb01) functionalised particles bound to E. coli MC1061 expressing mScarlet and naturally expressing OmpA-short. c) Nanobody 41 (Nb41) functionalised particles bound to E. coli MC1061 Δ ompA expressing mNeonGreen and OmpA-long. Scale bar = 5 μm.

Article Snippet: For E. coli , we developed chromosomally fluorescent strains expressing mNeonGreen or mScarlet proteins in E. coli MC1061 (ATCC 37493, obtained from the lab of Markus Seeger).

Techniques: Bacteria, Binding Assay, Imaging, Fluorescence, Expressing

Journal: Lab on a Chip

Article Title: Controlling spatial structure in minimal microbial communities by sequential capillary assembly

doi: 10.1039/d6lc00040a

Figure Lengend Snippet: Single species growth upon bacterial binding to sCAPA deposited particles. a) Graphical illustration of deposition and binding assay. Particles are first deposited with sCAPA, and the template is then transferred to a Petri dish and filled with BSA to cover the PDMS. Bacteria are then added to bind to the particles. Unbound bacteria are washed out with fresh PBS using a pipette. PBS is then replaced with agar media, before placing the petri dish under a microscope to image cell growth. b) Representative fluorescence images of S. aureus JE2 GFP bound to SbF1 deposited particles. i) Cells bound at t = 40 min after adding tryptone soy agar. ii) Cells growing after t = 140 min. iii) Cells growing at t = 260 min where colonies begin to merge. Scale bar = 50 μm. c) E. coli MC1061 mScarlet bound to Nb01 deposited particles. i) Cy3 image of cell binding at t = 40 min. ii) Phase contrast image of cell growth after t = 260 min. Cy3 fluorescence not visible at exponential phase due to weak fluorescence. iii) Cell growth at t = 310 min where colonies begin to merge. Scale bar = 50 μm. d) Binarised images in c used during image quantification. Binarised colony in white, red circles represent a circle with an equivalent area as overlaying colony to which a ‘colony radius’ is attributed. i) Cells bound at t = 40 min after adding tryptone soy agar, ii) colony formation after 260 min growth, iii) colonies begin to merge at 310 min. e) Quantification of the fraction of traps with observed microbial growth. Bars and errors represent mean and standard error of the mean, data points represent different fractions measured for each field of view for 3 separate templates for each bacterial strain. f) Distribution of number of bacteria bound in each trap as determined by particle localisation image analysis on ×60 magnification images of templates in PBS. Despite the particles all being the same size, the average number of bound bacteria to each particle notably varies. Mean number of bound bacteria and standard error of the mean displayed in top right. g) Distribution in time taken for individual colonies to reach a radius of 15 μm. Data points represent single growing colonies. Distributions represent all data for biological triplicates. Black lines represent mean and variance. h) Growth of indicated bacterial strains in liquid tryptone soy broth measured by optical density at 600 nm. Error bars represent standard error of the mean of 3 biological repeats and the black dotted line represents fit to a Gompertz growth law.

Article Snippet: For E. coli , we developed chromosomally fluorescent strains expressing mNeonGreen or mScarlet proteins in E. coli MC1061 (ATCC 37493, obtained from the lab of Markus Seeger).

Techniques: Binding Assay, Bacteria, Transferring, Microscopy, Fluorescence

Journal: Lab on a Chip

Article Title: Controlling spatial structure in minimal microbial communities by sequential capillary assembly

doi: 10.1039/d6lc00040a

Figure Lengend Snippet: Cell binding of microbial pairs with designed spatial structure. a–c) Representative images of selectively bound and growing cells across different time points. a) E. coli mScarlet (OmpA-short) and E. coli mNeonGreen (OmpA-long) in a sodium chloride lattice structure corresponding to Moran's I = −1. b) E. coli mScarlet (OmpA-short) and E. coli mNeonGreen (OmpA-long) in 8 × 8 patchy lattice structure corresponding to Moran's I = 0.75. c) S. aureus GFP and E. coli mScarlet (OmpA-long) in 1 : 24 ratio square lattice. i) Merged Cy3 and FITC image in PBS media, ii) cell growth after exchanging PBS with tryptone soy agar and growing at 37 °C. Distance between traps in all lattices is 25 μm. Time stamp in hours:mins after exchanging PBS for agar. Scale bar = 50 μm. d) Comparison of ideal vs. measured cell binding for spatial structures in a–c. Bars represent mean of 4 independent templates. e) Fraction of growing cells for sum of green and red cells. Data points represent measured growing fraction in individual microscope fields of view, for 4 independent templates. Error bars represent standard error of the mean.

Article Snippet: For E. coli , we developed chromosomally fluorescent strains expressing mNeonGreen or mScarlet proteins in E. coli MC1061 (ATCC 37493, obtained from the lab of Markus Seeger).

Techniques: Binding Assay, Comparison, Microscopy

Journal: Lab on a Chip

Article Title: Controlling spatial structure in minimal microbial communities by sequential capillary assembly

doi: 10.1039/d6lc00040a

Figure Lengend Snippet: Nanobody-functionalised silica particles bind selectively to target bacteria. i) Graphical illustration of particle–bacteria binding strategy. The cysteine group on the nanobody is bound to a PEG11 linker via a maleimide bond which, in turn, is bound to streptavidin-functionalised 2.7 μm silica particles via a biotin bond. ii) Particle–bacteria binding after mixing for 30 min in a 1.5 ml Eppendorf and imaging in a 96-well plate in the phase contrast channel or iii) fluorescence channel. White arrow highlights a silica particle, blue arrow highlights a bacterium. a) Sybody-F1 (SbF1) functionalised particles bound to S. aureus GFP. b) Nanobody 01 (Nb01) functionalised particles bound to E. coli MC1061 expressing mScarlet and naturally expressing OmpA-short. c) Nanobody 41 (Nb41) functionalised particles bound to E. coli MC1061 Δ ompA expressing mNeonGreen and OmpA-long. Scale bar = 5 μm.

Article Snippet: For E. coli , we developed chromosomally fluorescent strains expressing mNeonGreen or mScarlet proteins in E. coli MC1061 (ATCC 37493, obtained from the lab of Markus Seeger).

Techniques: Bacteria, Binding Assay, Imaging, Fluorescence, Expressing

Journal: Lab on a Chip

Article Title: Controlling spatial structure in minimal microbial communities by sequential capillary assembly

doi: 10.1039/d6lc00040a

Figure Lengend Snippet: Single species growth upon bacterial binding to sCAPA deposited particles. a) Graphical illustration of deposition and binding assay. Particles are first deposited with sCAPA, and the template is then transferred to a Petri dish and filled with BSA to cover the PDMS. Bacteria are then added to bind to the particles. Unbound bacteria are washed out with fresh PBS using a pipette. PBS is then replaced with agar media, before placing the petri dish under a microscope to image cell growth. b) Representative fluorescence images of S. aureus JE2 GFP bound to SbF1 deposited particles. i) Cells bound at t = 40 min after adding tryptone soy agar. ii) Cells growing after t = 140 min. iii) Cells growing at t = 260 min where colonies begin to merge. Scale bar = 50 μm. c) E. coli MC1061 mScarlet bound to Nb01 deposited particles. i) Cy3 image of cell binding at t = 40 min. ii) Phase contrast image of cell growth after t = 260 min. Cy3 fluorescence not visible at exponential phase due to weak fluorescence. iii) Cell growth at t = 310 min where colonies begin to merge. Scale bar = 50 μm. d) Binarised images in c used during image quantification. Binarised colony in white, red circles represent a circle with an equivalent area as overlaying colony to which a ‘colony radius’ is attributed. i) Cells bound at t = 40 min after adding tryptone soy agar, ii) colony formation after 260 min growth, iii) colonies begin to merge at 310 min. e) Quantification of the fraction of traps with observed microbial growth. Bars and errors represent mean and standard error of the mean, data points represent different fractions measured for each field of view for 3 separate templates for each bacterial strain. f) Distribution of number of bacteria bound in each trap as determined by particle localisation image analysis on ×60 magnification images of templates in PBS. Despite the particles all being the same size, the average number of bound bacteria to each particle notably varies. Mean number of bound bacteria and standard error of the mean displayed in top right. g) Distribution in time taken for individual colonies to reach a radius of 15 μm. Data points represent single growing colonies. Distributions represent all data for biological triplicates. Black lines represent mean and variance. h) Growth of indicated bacterial strains in liquid tryptone soy broth measured by optical density at 600 nm. Error bars represent standard error of the mean of 3 biological repeats and the black dotted line represents fit to a Gompertz growth law.

Article Snippet: For E. coli , we developed chromosomally fluorescent strains expressing mNeonGreen or mScarlet proteins in E. coli MC1061 (ATCC 37493, obtained from the lab of Markus Seeger).

Techniques: Binding Assay, Bacteria, Transferring, Microscopy, Fluorescence

Journal: Lab on a Chip

Article Title: Controlling spatial structure in minimal microbial communities by sequential capillary assembly

doi: 10.1039/d6lc00040a

Figure Lengend Snippet: Cell binding of microbial pairs with designed spatial structure. a–c) Representative images of selectively bound and growing cells across different time points. a) E. coli mScarlet (OmpA-short) and E. coli mNeonGreen (OmpA-long) in a sodium chloride lattice structure corresponding to Moran's I = −1. b) E. coli mScarlet (OmpA-short) and E. coli mNeonGreen (OmpA-long) in 8 × 8 patchy lattice structure corresponding to Moran's I = 0.75. c) S. aureus GFP and E. coli mScarlet (OmpA-long) in 1 : 24 ratio square lattice. i) Merged Cy3 and FITC image in PBS media, ii) cell growth after exchanging PBS with tryptone soy agar and growing at 37 °C. Distance between traps in all lattices is 25 μm. Time stamp in hours:mins after exchanging PBS for agar. Scale bar = 50 μm. d) Comparison of ideal vs. measured cell binding for spatial structures in a–c. Bars represent mean of 4 independent templates. e) Fraction of growing cells for sum of green and red cells. Data points represent measured growing fraction in individual microscope fields of view, for 4 independent templates. Error bars represent standard error of the mean.

Article Snippet: For E. coli , we developed chromosomally fluorescent strains expressing mNeonGreen or mScarlet proteins in E. coli MC1061 (ATCC 37493, obtained from the lab of Markus Seeger).

Techniques: Binding Assay, Comparison, Microscopy

Journal: NPJ Regenerative Medicine

Article Title: Bioengineered chondrocyte-products from human induced pluripotent stem cells are useful for repairing articular cartilage injury in minipig model

doi: 10.1038/s41536-025-00420-3

Figure Lengend Snippet: A Schematic representation of the protocol for fabricating cartilaginous particles from ExpLBM cells. B , C Fabrication of ExpLBM-derived cartilaginous particles in 96-well cell culture dish. D Histology of ExpLBM-derived cartilaginous particle. Safranin O-fast green-iron hematoxylin staining (Safranin O). Scale bars: 500 μm. E Immunofluorescence examination of cartilaginous particle. Semi-serial sections were immunostained for COL2, SOX9, and COL10. Scale bars: 500 μm. F qPCR analysis for COL2A1, SOX9, and IHH expression in cartilage particles. All values were normalized to ACTB mRNA levels ( n = 3). Data are presented as the means ± SEMs. * p < 0.05.

Article Snippet: The antibodies used were as follows: anti-hVimentin (catalog no. 10515; Progen Biotechnik), COL2 (catalog no. MA1-37493; Thermo Fisher Scientific), SOX9 (catalog no. AB5535; Sigma-Aldrich), COL10 (catalog no. 14-9771-80; Thermo Fisher Scientific), aggrecan (catalog no. 13880-1-AP; Proteintech), and Alexa-647 and Alexa-488 (Thermo Fisher Scientific).

Techniques: Derivative Assay, Cell Culture, Staining, Immunofluorescence, Expressing

Journal: NPJ Regenerative Medicine

Article Title: Bioengineered chondrocyte-products from human induced pluripotent stem cells are useful for repairing articular cartilage injury in minipig model

doi: 10.1038/s41536-025-00420-3

Figure Lengend Snippet: A Schematic of the transplantation of ExpLBM-derived cartilaginous particles in a minipig model. B Gross appearance of the joint surface before (left) and after (right) transplantation during surgery. C Gross appearance of the joint surface at 2 weeks postoperatively. D Histological examination of the repaired osteochondral defect and sham. Semi-serial sections were stained for hematoxylin–eosin and Safranin O. Scale bars: 500 μm. E Immunofluorescence examination of the repaired osteochondral defect and sham. Samples were harvested 2 weeks after transplantation. Semi-serial sections were immunostained for hVimentin, Aggrecan, COL2, SOX9, and COL10. Scale bars: 500 μm. F Histological analysis based on the ICRS scoring system ( n = 4 knees of minipigs in chondro-particles transplants group, and n = 6 in sham group). Statistical significance was determined using unpaired one-way ANOVA with Tukey’s post hoc analysis. *** p < 0.001.

Article Snippet: The antibodies used were as follows: anti-hVimentin (catalog no. 10515; Progen Biotechnik), COL2 (catalog no. MA1-37493; Thermo Fisher Scientific), SOX9 (catalog no. AB5535; Sigma-Aldrich), COL10 (catalog no. 14-9771-80; Thermo Fisher Scientific), aggrecan (catalog no. 13880-1-AP; Proteintech), and Alexa-647 and Alexa-488 (Thermo Fisher Scientific).

Techniques: Transplantation Assay, Derivative Assay, Staining, Immunofluorescence

Journal: NPJ Regenerative Medicine

Article Title: Bioengineered chondrocyte-products from human induced pluripotent stem cells are useful for repairing articular cartilage injury in minipig model

doi: 10.1038/s41536-025-00420-3

Figure Lengend Snippet: A Schematic representation of the protocol for fabricating cartilaginous plate from ExpLBM cells. B Gross appearance of the fabricated ExpLBM-derived cartilaginous plate. C Histology of ExpLBM-derived cartilaginous plate. Safranin O staining. Scale bars: 500 μm. D Immunofluorescence examination of cartilaginous plate. Semi-serial sections were immunostained for COL2, SOX9, and COL10. Scale bars: 500 μm. E qPCR analysis for COL2A1, SOX9, and IHH expression in cartilage plate. All values were normalized to ACTB mRNA levels ( n = 3). Data are presented as the means ± SEMs. * p < 0.05.

Article Snippet: The antibodies used were as follows: anti-hVimentin (catalog no. 10515; Progen Biotechnik), COL2 (catalog no. MA1-37493; Thermo Fisher Scientific), SOX9 (catalog no. AB5535; Sigma-Aldrich), COL10 (catalog no. 14-9771-80; Thermo Fisher Scientific), aggrecan (catalog no. 13880-1-AP; Proteintech), and Alexa-647 and Alexa-488 (Thermo Fisher Scientific).

Techniques: Derivative Assay, Staining, Immunofluorescence, Expressing

Journal: NPJ Regenerative Medicine

Article Title: Bioengineered chondrocyte-products from human induced pluripotent stem cells are useful for repairing articular cartilage injury in minipig model

doi: 10.1038/s41536-025-00420-3

Figure Lengend Snippet: A Schematic of the transplantation of ExpLBM-derived cartilaginous plate in a minipig model. B Gross appearance of the joint surface before (left) and after (right) transplantation during surgery. C Gross appearance of the joint surface at 2 weeks postoperatively. D Histological examination of the repaired osteochondral defect and sham. Semi-serial sections were stained for hematoxylin–eosin and Safranin O. Scale bars: 500 μm. E Immunohistochemical examination of the repaired osteochondral defect and sham. Semi-serial sections were immunostained for hVimentin, Aggrecan, COL2, SOX9, and COL10. Scale bars: 500 μm. F Histological analysis based on the ICRS scoring system (n = 6 knees of minipigs in cartilaginous plate transplants group, and n = 6 in sham group). Statistical significance was determined using unpaired one-way ANOVA with Tukey’s post hoc analysis. *** p < 0.001.

Article Snippet: The antibodies used were as follows: anti-hVimentin (catalog no. 10515; Progen Biotechnik), COL2 (catalog no. MA1-37493; Thermo Fisher Scientific), SOX9 (catalog no. AB5535; Sigma-Aldrich), COL10 (catalog no. 14-9771-80; Thermo Fisher Scientific), aggrecan (catalog no. 13880-1-AP; Proteintech), and Alexa-647 and Alexa-488 (Thermo Fisher Scientific).

Techniques: Transplantation Assay, Derivative Assay, Staining, Immunohistochemical staining