Structured Review

Millipore proteins bovine serum albumin bsa
Quantitative peptidomics scheme for the characterization of rhCPD substrate specificity using the tryptic peptide library and representative spectra. (A) Quantitative peptidomics scheme. Tryptic peptides were obtained from digestion of five selected proteins <t>(BSA,</t> bovine thyroglobulin, bovine <t>α-lactalbumin</t> and human α and β–hemoglobin) with trypsin. The resultant peptide library was aliquoted and digested with no enzyme or different rhCPD concentrations of 0.1, 1, 10, and 100 nM for 16 h at 37°C. After incubation samples were labeled with one of five stable isotopic TMAB-NHS tags (D0 = 100 nM; D3 = 10 nM; D6 = 1 nM; D9 = 0.1 nM; D12 = No enzyme). Then, samples were pooled and analyzed by LC-MS. Examples of representative data are shown for (B) non-substrates, (C) good substrates, (D) weak substrates and (E) products.
Proteins Bovine Serum Albumin Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains"

Article Title: Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains

Journal: PLoS ONE

doi: 10.1371/journal.pone.0187778

Quantitative peptidomics scheme for the characterization of rhCPD substrate specificity using the tryptic peptide library and representative spectra. (A) Quantitative peptidomics scheme. Tryptic peptides were obtained from digestion of five selected proteins (BSA, bovine thyroglobulin, bovine α-lactalbumin and human α and β–hemoglobin) with trypsin. The resultant peptide library was aliquoted and digested with no enzyme or different rhCPD concentrations of 0.1, 1, 10, and 100 nM for 16 h at 37°C. After incubation samples were labeled with one of five stable isotopic TMAB-NHS tags (D0 = 100 nM; D3 = 10 nM; D6 = 1 nM; D9 = 0.1 nM; D12 = No enzyme). Then, samples were pooled and analyzed by LC-MS. Examples of representative data are shown for (B) non-substrates, (C) good substrates, (D) weak substrates and (E) products.
Figure Legend Snippet: Quantitative peptidomics scheme for the characterization of rhCPD substrate specificity using the tryptic peptide library and representative spectra. (A) Quantitative peptidomics scheme. Tryptic peptides were obtained from digestion of five selected proteins (BSA, bovine thyroglobulin, bovine α-lactalbumin and human α and β–hemoglobin) with trypsin. The resultant peptide library was aliquoted and digested with no enzyme or different rhCPD concentrations of 0.1, 1, 10, and 100 nM for 16 h at 37°C. After incubation samples were labeled with one of five stable isotopic TMAB-NHS tags (D0 = 100 nM; D3 = 10 nM; D6 = 1 nM; D9 = 0.1 nM; D12 = No enzyme). Then, samples were pooled and analyzed by LC-MS. Examples of representative data are shown for (B) non-substrates, (C) good substrates, (D) weak substrates and (E) products.

Techniques Used: Incubation, Labeling, Liquid Chromatography with Mass Spectroscopy

2) Product Images from "Tetraphenylporphyrin as a protein label for triple detection analytical systems"

Article Title: Tetraphenylporphyrin as a protein label for triple detection analytical systems

Journal: Heliyon

doi: 10.1016/j.heliyon.2015.e00053

Differential pulse voltammograms representing oxidation of 0.1 mM Tpp in the presence of proteins: BSA, CEA and IgG (4 mg·mL −1 ) obtained on glassy carbon electrode in the DMSO solution containing 0.01 M tetraoctylammonium bromide.
Figure Legend Snippet: Differential pulse voltammograms representing oxidation of 0.1 mM Tpp in the presence of proteins: BSA, CEA and IgG (4 mg·mL −1 ) obtained on glassy carbon electrode in the DMSO solution containing 0.01 M tetraoctylammonium bromide.

Techniques Used:

3) Product Images from "Development of an aptamer-based affinity purification method for vascular endothelial growth factor"

Article Title: Development of an aptamer-based affinity purification method for vascular endothelial growth factor

Journal: Biotechnology Reports

doi: 10.1016/j.btre.2015.08.006

Characterization of aptamer target binding via microscale thermophoresis and electrophoresis mobility shift assay (EMSA). Left: saturation curve of V7t1 aptamer and VEGF. Cy3-labeled V7t1 was incubated with different concentrations of VEGF and afterwards analyzed by microscale thermophoresis as three-fold replicates. A K D of 75.9 nM ± 13.0 nM was determined for this interaction. Right: analysis of aptamer binding to the target protein VEGF and the non-target proteins myoglobin (Myo), α-chymotrypsin (Chy) and bovine serum albumin (BSA) via EMSA. The Cy3-labeled aptamer V7t1 was incubated with an excess of protein, respectively. Afterwards the samples were analyzed by agarose gel electrophoresis. Pure VEGF and V7t1 were used as negative controls, respectively. Results of the EMSA demonstrate a specific binding of V7t1 to VEGF in comparison to the other tested proteins.
Figure Legend Snippet: Characterization of aptamer target binding via microscale thermophoresis and electrophoresis mobility shift assay (EMSA). Left: saturation curve of V7t1 aptamer and VEGF. Cy3-labeled V7t1 was incubated with different concentrations of VEGF and afterwards analyzed by microscale thermophoresis as three-fold replicates. A K D of 75.9 nM ± 13.0 nM was determined for this interaction. Right: analysis of aptamer binding to the target protein VEGF and the non-target proteins myoglobin (Myo), α-chymotrypsin (Chy) and bovine serum albumin (BSA) via EMSA. The Cy3-labeled aptamer V7t1 was incubated with an excess of protein, respectively. Afterwards the samples were analyzed by agarose gel electrophoresis. Pure VEGF and V7t1 were used as negative controls, respectively. Results of the EMSA demonstrate a specific binding of V7t1 to VEGF in comparison to the other tested proteins.

Techniques Used: Binding Assay, Microscale Thermophoresis, Electrophoresis, Mobility Shift, Labeling, Incubation, Agarose Gel Electrophoresis

4) Product Images from "Development of an aptamer-based affinity purification method for vascular endothelial growth factor"

Article Title: Development of an aptamer-based affinity purification method for vascular endothelial growth factor

Journal: Biotechnology Reports

doi: 10.1016/j.btre.2015.08.006

Characterization of aptamer target binding via microscale thermophoresis and electrophoresis mobility shift assay (EMSA). Left: saturation curve of V7t1 aptamer and VEGF. Cy3-labeled V7t1 was incubated with different concentrations of VEGF and afterwards analyzed by microscale thermophoresis as three-fold replicates. A K D of 75.9 nM ± 13.0 nM was determined for this interaction. Right: analysis of aptamer binding to the target protein VEGF and the non-target proteins myoglobin (Myo), α-chymotrypsin (Chy) and bovine serum albumin (BSA) via EMSA. The Cy3-labeled aptamer V7t1 was incubated with an excess of protein, respectively. Afterwards the samples were analyzed by agarose gel electrophoresis. Pure VEGF and V7t1 were used as negative controls, respectively. Results of the EMSA demonstrate a specific binding of V7t1 to VEGF in comparison to the other tested proteins.
Figure Legend Snippet: Characterization of aptamer target binding via microscale thermophoresis and electrophoresis mobility shift assay (EMSA). Left: saturation curve of V7t1 aptamer and VEGF. Cy3-labeled V7t1 was incubated with different concentrations of VEGF and afterwards analyzed by microscale thermophoresis as three-fold replicates. A K D of 75.9 nM ± 13.0 nM was determined for this interaction. Right: analysis of aptamer binding to the target protein VEGF and the non-target proteins myoglobin (Myo), α-chymotrypsin (Chy) and bovine serum albumin (BSA) via EMSA. The Cy3-labeled aptamer V7t1 was incubated with an excess of protein, respectively. Afterwards the samples were analyzed by agarose gel electrophoresis. Pure VEGF and V7t1 were used as negative controls, respectively. Results of the EMSA demonstrate a specific binding of V7t1 to VEGF in comparison to the other tested proteins.

Techniques Used: Binding Assay, Microscale Thermophoresis, Electrophoresis, Mobility Shift, Labeling, Incubation, Agarose Gel Electrophoresis

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    Millipore fibronectin bovine plasma
    ERK signaling activity in composite tissues depends on interface boundary conditions. a . Confocal immunofluorescence for phosphorylated ERK (pERK) and E-cadherin in “interior mesenchyme” and “interior epithelium” tissues and intensity-colored images of pERK staining. b . Radial quantification of normalized pERK intensity relative to inner tissue radius for each pattern type. Vertical dashed line marks the tissue interface position (r = 1.0 a.u.), ribbons are mean ± standard deviation (s.d.) of normalized pERK intensity for n = 10 interior epithelium and n = 15 interior mesenchyme tissues pooled from two independent experiments. Cells were patterned on 7.5%/0.25% (Am/Bis) BP-PA hydrogels sequentially photopatterned with polyT 20 G and polyT 20 F ssDNA (t = 90-120 s exposure at 254 nm) and subsequently functionalized with 20 μg ml -1 <t>fibronectin.</t> All tissues were patterned as described in figure 4 and kept in culture for 12-15 hours to permit interface formation before fixation and staining.
    Fibronectin Bovine Plasma, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore fibronectin
    ERK signaling activity in composite tissues depends on interface boundary conditions. a . Confocal immunofluorescence for phosphorylated ERK (pERK) and E-cadherin in “interior mesenchyme” and “interior epithelium” tissues and intensity-colored images of pERK staining. b . Radial quantification of normalized pERK intensity relative to inner tissue radius for each pattern type. Vertical dashed line marks the tissue interface position (r = 1.0 a.u.), ribbons are mean ± standard deviation (s.d.) of normalized pERK intensity for n = 10 interior epithelium and n = 15 interior mesenchyme tissues pooled from two independent experiments. Cells were patterned on 7.5%/0.25% (Am/Bis) BP-PA hydrogels sequentially photopatterned with polyT 20 G and polyT 20 F ssDNA (t = 90-120 s exposure at 254 nm) and subsequently functionalized with 20 μg ml -1 <t>fibronectin.</t> All tissues were patterned as described in figure 4 and kept in culture for 12-15 hours to permit interface formation before fixation and staining.
    Fibronectin, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fibronectin/product/Millipore
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    fibronectin - by Bioz Stars, 2022-11
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    Image Search Results


    ERK signaling activity in composite tissues depends on interface boundary conditions. a . Confocal immunofluorescence for phosphorylated ERK (pERK) and E-cadherin in “interior mesenchyme” and “interior epithelium” tissues and intensity-colored images of pERK staining. b . Radial quantification of normalized pERK intensity relative to inner tissue radius for each pattern type. Vertical dashed line marks the tissue interface position (r = 1.0 a.u.), ribbons are mean ± standard deviation (s.d.) of normalized pERK intensity for n = 10 interior epithelium and n = 15 interior mesenchyme tissues pooled from two independent experiments. Cells were patterned on 7.5%/0.25% (Am/Bis) BP-PA hydrogels sequentially photopatterned with polyT 20 G and polyT 20 F ssDNA (t = 90-120 s exposure at 254 nm) and subsequently functionalized with 20 μg ml -1 fibronectin. All tissues were patterned as described in figure 4 and kept in culture for 12-15 hours to permit interface formation before fixation and staining.

    Journal: bioRxiv

    Article Title: Independent control over cell patterning and adhesion on hydrogel substrates for tissue interface mechanobiology

    doi: 10.1101/2022.11.16.516785

    Figure Lengend Snippet: ERK signaling activity in composite tissues depends on interface boundary conditions. a . Confocal immunofluorescence for phosphorylated ERK (pERK) and E-cadherin in “interior mesenchyme” and “interior epithelium” tissues and intensity-colored images of pERK staining. b . Radial quantification of normalized pERK intensity relative to inner tissue radius for each pattern type. Vertical dashed line marks the tissue interface position (r = 1.0 a.u.), ribbons are mean ± standard deviation (s.d.) of normalized pERK intensity for n = 10 interior epithelium and n = 15 interior mesenchyme tissues pooled from two independent experiments. Cells were patterned on 7.5%/0.25% (Am/Bis) BP-PA hydrogels sequentially photopatterned with polyT 20 G and polyT 20 F ssDNA (t = 90-120 s exposure at 254 nm) and subsequently functionalized with 20 μg ml -1 fibronectin. All tissues were patterned as described in figure 4 and kept in culture for 12-15 hours to permit interface formation before fixation and staining.

    Article Snippet: Hydrogels were then partially dried and a plastic clip-on 8-well slide with a silicone gasket (CCS-8, MatTek) was assembled around the region containing photopatterned ssDNA features and wells were incubated in 10-20 μg ml-1 bovine serum fibronectin (1 mg ml-1, F1141, Sigma) diluted in sterile 50 mM HEPES (pH 8.5) overnight at 4°C.

    Techniques: Activity Assay, Immunofluorescence, Staining, Standard Deviation

    Epithelial-mesenchymal tissue interfaces show emergent organization that depends on initial tissue geometry. a . Schematic of two-part ssDNA patterning design consisting of concentric circles. b . Example images of co-patterned H2B-iRFP 3T3s and H2B-Venus MDCKs acquired starting 1 hour after patterning and every 2 hours for 10 hours total. c-d . Inner tissue area and circularity measured from n = 8 interior mesenchyme and n = 9 interior epithelium tissues from an example experiment. e-f . Summary plots of tissue area and circularity at time of fixation (t = 12-15 hrs after patterning) measured for n = 11 interior mesenchyme and n = 15 interior epithelium tissues collected from two independent replicates. Individual experiment means (markers with black borders) are offset to the right of each group and data points for each experiment are organized by shape. p-values for each replicate are computed using Welch’s two-sided t-test. Dashed horizontal line in panels c and e represents the inner tissue patterning radius (r = 175 μm). g . Confocal micrograph of an “interior mesenchyme” microtissue stained for F-actin and E-cadherin. Inset , composite and single channel images show alignment of epithelial cells and fibroblasts at the interface (dashed lines in each panel). h . Radial quantification of normalized E-cadherin and F-actin fluorescence intensity relative to inner tissue radius. i . Example “interior epithelium” tissue stained for E-cadherin and F-actin and j . radial quantification of normalized fluorescence intensity relative to inner tissue radius. Vertical dashed line marks the tissue interface position (r = 1.0 a.u.), ribbons are mean ± s.d. of tissues analyzed in panel e . In all experiments, MDCK cells are patterned using the G/G’ ssDNA pair, while 3T3 cells are patterned using the F/F’ ssDNA pair. Cells were patterned on 7.5%/0.25% (Am/Bis) BP-PA hydrogels sequentially photopatterned with polyT 20 G and polyT 20 F ssDNA (t = 90 s exposure at 254 nm each) and functionalized with 20 μg ml -1 fibronectin. See also: figure S4-S5 and movies S2-S3 .

    Journal: bioRxiv

    Article Title: Independent control over cell patterning and adhesion on hydrogel substrates for tissue interface mechanobiology

    doi: 10.1101/2022.11.16.516785

    Figure Lengend Snippet: Epithelial-mesenchymal tissue interfaces show emergent organization that depends on initial tissue geometry. a . Schematic of two-part ssDNA patterning design consisting of concentric circles. b . Example images of co-patterned H2B-iRFP 3T3s and H2B-Venus MDCKs acquired starting 1 hour after patterning and every 2 hours for 10 hours total. c-d . Inner tissue area and circularity measured from n = 8 interior mesenchyme and n = 9 interior epithelium tissues from an example experiment. e-f . Summary plots of tissue area and circularity at time of fixation (t = 12-15 hrs after patterning) measured for n = 11 interior mesenchyme and n = 15 interior epithelium tissues collected from two independent replicates. Individual experiment means (markers with black borders) are offset to the right of each group and data points for each experiment are organized by shape. p-values for each replicate are computed using Welch’s two-sided t-test. Dashed horizontal line in panels c and e represents the inner tissue patterning radius (r = 175 μm). g . Confocal micrograph of an “interior mesenchyme” microtissue stained for F-actin and E-cadherin. Inset , composite and single channel images show alignment of epithelial cells and fibroblasts at the interface (dashed lines in each panel). h . Radial quantification of normalized E-cadherin and F-actin fluorescence intensity relative to inner tissue radius. i . Example “interior epithelium” tissue stained for E-cadherin and F-actin and j . radial quantification of normalized fluorescence intensity relative to inner tissue radius. Vertical dashed line marks the tissue interface position (r = 1.0 a.u.), ribbons are mean ± s.d. of tissues analyzed in panel e . In all experiments, MDCK cells are patterned using the G/G’ ssDNA pair, while 3T3 cells are patterned using the F/F’ ssDNA pair. Cells were patterned on 7.5%/0.25% (Am/Bis) BP-PA hydrogels sequentially photopatterned with polyT 20 G and polyT 20 F ssDNA (t = 90 s exposure at 254 nm each) and functionalized with 20 μg ml -1 fibronectin. See also: figure S4-S5 and movies S2-S3 .

    Article Snippet: Hydrogels were then partially dried and a plastic clip-on 8-well slide with a silicone gasket (CCS-8, MatTek) was assembled around the region containing photopatterned ssDNA features and wells were incubated in 10-20 μg ml-1 bovine serum fibronectin (1 mg ml-1, F1141, Sigma) diluted in sterile 50 mM HEPES (pH 8.5) overnight at 4°C.

    Techniques: Staining, Fluorescence

    Photopatterned BP-PA hydrogel mechanics, fibroblast spreading, and focal adhesion formation are comparable to control BP-PA gels. a . Schematic of hydrogel UV exposure and microindentation with example force vs. indentation depth curve obtained for a 255 μm diameter cylindrical indenter on a 3%/0.05% Am/Bis ratio BP-PA hydrogel. Hydrogels were incubated in ssDNA (200 μM polyT 20 G) and exposed to 254 nm light for 90 seconds through one half of a quartz slide (+UV), with the other side blocked from UV exposure (control). b . Quantification of E for BP-PA hydrogels cast with 3-7.5% Am, 0.01-0.25% Bis, and 3 mM BPMAC (see table S2 for mean ± s.d. for n = 2-4 hydrogels per Am/Bis composition). Individual data points are identified by shape, bar heights represent overall mean. c . 3T3 fibroblasts spreading on +UV and control hydrogel regions functionalized with 20 μg ml -1 fibronectin. Cells adhered for 16-24 hours prior to fixation and staining for F-actin and nuclei (DAPI). d . Quantification of 3T3 spread area on control and +UV hydrogel regions from BP-PA hydrogels cast with varying Am/Bis compositions; control, n = 108, 117, 116, 119, 110 cells and +UV, n = 92, 128, 129, 135, 110 cells. Data are pooled from two independent replicate experiments identified by marker shape, individual experiment means are overlaid onto distributions (black borders), p -value comparisons between groups are from two-sided Kolmogorov-Smirnov tests. e . 3T3 fibroblast adhered to a 7.5%/0.25% (Am/Bis) BP-PA hydrogel containing 250 μm circular ssDNA features (labeled with FAM_F’), nuclei (DAPI), F-actin, and focal adhesions (vinculin). Insets , detail on regions marked by dashed boxes. f . Quantification of L adhesion for cells on unexposed (off DNA) and UV-exposed (on DNA) hydrogel regions. Vertical dashed lines represent the mean of n = 94 (off DNA) and 87 (on DNA) focal adhesions measured from n = 29 cells pooled from two independent experiments (biological replicates). p -value was computed using a two-sided Kolmogorov-Smirnov test.

    Journal: bioRxiv

    Article Title: Independent control over cell patterning and adhesion on hydrogel substrates for tissue interface mechanobiology

    doi: 10.1101/2022.11.16.516785

    Figure Lengend Snippet: Photopatterned BP-PA hydrogel mechanics, fibroblast spreading, and focal adhesion formation are comparable to control BP-PA gels. a . Schematic of hydrogel UV exposure and microindentation with example force vs. indentation depth curve obtained for a 255 μm diameter cylindrical indenter on a 3%/0.05% Am/Bis ratio BP-PA hydrogel. Hydrogels were incubated in ssDNA (200 μM polyT 20 G) and exposed to 254 nm light for 90 seconds through one half of a quartz slide (+UV), with the other side blocked from UV exposure (control). b . Quantification of E for BP-PA hydrogels cast with 3-7.5% Am, 0.01-0.25% Bis, and 3 mM BPMAC (see table S2 for mean ± s.d. for n = 2-4 hydrogels per Am/Bis composition). Individual data points are identified by shape, bar heights represent overall mean. c . 3T3 fibroblasts spreading on +UV and control hydrogel regions functionalized with 20 μg ml -1 fibronectin. Cells adhered for 16-24 hours prior to fixation and staining for F-actin and nuclei (DAPI). d . Quantification of 3T3 spread area on control and +UV hydrogel regions from BP-PA hydrogels cast with varying Am/Bis compositions; control, n = 108, 117, 116, 119, 110 cells and +UV, n = 92, 128, 129, 135, 110 cells. Data are pooled from two independent replicate experiments identified by marker shape, individual experiment means are overlaid onto distributions (black borders), p -value comparisons between groups are from two-sided Kolmogorov-Smirnov tests. e . 3T3 fibroblast adhered to a 7.5%/0.25% (Am/Bis) BP-PA hydrogel containing 250 μm circular ssDNA features (labeled with FAM_F’), nuclei (DAPI), F-actin, and focal adhesions (vinculin). Insets , detail on regions marked by dashed boxes. f . Quantification of L adhesion for cells on unexposed (off DNA) and UV-exposed (on DNA) hydrogel regions. Vertical dashed lines represent the mean of n = 94 (off DNA) and 87 (on DNA) focal adhesions measured from n = 29 cells pooled from two independent experiments (biological replicates). p -value was computed using a two-sided Kolmogorov-Smirnov test.

    Article Snippet: Hydrogels were then partially dried and a plastic clip-on 8-well slide with a silicone gasket (CCS-8, MatTek) was assembled around the region containing photopatterned ssDNA features and wells were incubated in 10-20 μg ml-1 bovine serum fibronectin (1 mg ml-1, F1141, Sigma) diluted in sterile 50 mM HEPES (pH 8.5) overnight at 4°C.

    Techniques: Incubation, Staining, Marker, Labeling

    Cell capture and long-term adhesion on polyacrylamide substrates through orthogonal ssDNA and ECM patterning. a . Schematic of the top , ssDNA photolithography and BP-PA gel surface functionalization with fibronectin, and bottom , capture of lipid-ssDNA labeled cells. Cell labeling with lipid-ssDNA and sequence-matched “handle” ssDNA enables specific cell capture to hydrogel-bound ssDNA patterns. Captured cells subsequently adhere to the substrate using fibronectin ligands. b . Brightfield ( top ) and fluorescence ( bottom ) images of MDCK cells captured on a 500 μm diameter circular ssDNA feature. c . Quantification of MDCK cell capture (#) on 50, 100, 200, and 500 μm diameter ssDNA circles. Data depict n = 9 features per ssDNA diameter per three independent replicate experiments ( n = 27 features total per ssDNA diameter) and means of each experiment (black borders). Experiment means are shifted +30 μm along the +x axis for clarity. d . Top , maximum intensity projection ( xy ) of photopatterned ssDNA (polyT 20 F) and fibronectin on a BP-PA hydrogel from a 10x z-stack (2.5 μm per frame, 147.5 μm total height, 59 total frames). Bottom , maximum xz intensity across the highlighted 25 pixel region. d’ . Normalized mean fluorescence intensity sampled across the outlined portion of the xy image in panel d. e . Time lapse image sequences (10 hrs total) of cells adhering to circular, triangular, square, and star-shaped ssDNA features with fixed areas equivalent to a 200 μm diameter circle (A = 3.14×10 4 μm 2 ). e’ Inset , detail of the star-shaped pattern. All experiments were performed on 7.5%/0.25% (Am/Bis) BP-PA hydrogels photopatterned with 200 μM polyT 20 G ssDNA for 90 seconds and functionalized with 20 μg ml -1 fibronectin. Cells were labeled with lipid anchors and “G’ handle” ssDNA. Cells in panel b are labeled using CellTracker Deep Red and ssDNA in b and e is visualized using 2x SYBR Gold nucleic acid stain. Photopatterned polyT 20 F ssDNA in panels d-d’ is visualized with FAM_F’ ssDNA probe and fibronectin is visualized with a rabbit anti-fibronectin primary antibody and Alexa 647 secondary antibodies. See also: figure S1-S2 and movie S1 .

    Journal: bioRxiv

    Article Title: Independent control over cell patterning and adhesion on hydrogel substrates for tissue interface mechanobiology

    doi: 10.1101/2022.11.16.516785

    Figure Lengend Snippet: Cell capture and long-term adhesion on polyacrylamide substrates through orthogonal ssDNA and ECM patterning. a . Schematic of the top , ssDNA photolithography and BP-PA gel surface functionalization with fibronectin, and bottom , capture of lipid-ssDNA labeled cells. Cell labeling with lipid-ssDNA and sequence-matched “handle” ssDNA enables specific cell capture to hydrogel-bound ssDNA patterns. Captured cells subsequently adhere to the substrate using fibronectin ligands. b . Brightfield ( top ) and fluorescence ( bottom ) images of MDCK cells captured on a 500 μm diameter circular ssDNA feature. c . Quantification of MDCK cell capture (#) on 50, 100, 200, and 500 μm diameter ssDNA circles. Data depict n = 9 features per ssDNA diameter per three independent replicate experiments ( n = 27 features total per ssDNA diameter) and means of each experiment (black borders). Experiment means are shifted +30 μm along the +x axis for clarity. d . Top , maximum intensity projection ( xy ) of photopatterned ssDNA (polyT 20 F) and fibronectin on a BP-PA hydrogel from a 10x z-stack (2.5 μm per frame, 147.5 μm total height, 59 total frames). Bottom , maximum xz intensity across the highlighted 25 pixel region. d’ . Normalized mean fluorescence intensity sampled across the outlined portion of the xy image in panel d. e . Time lapse image sequences (10 hrs total) of cells adhering to circular, triangular, square, and star-shaped ssDNA features with fixed areas equivalent to a 200 μm diameter circle (A = 3.14×10 4 μm 2 ). e’ Inset , detail of the star-shaped pattern. All experiments were performed on 7.5%/0.25% (Am/Bis) BP-PA hydrogels photopatterned with 200 μM polyT 20 G ssDNA for 90 seconds and functionalized with 20 μg ml -1 fibronectin. Cells were labeled with lipid anchors and “G’ handle” ssDNA. Cells in panel b are labeled using CellTracker Deep Red and ssDNA in b and e is visualized using 2x SYBR Gold nucleic acid stain. Photopatterned polyT 20 F ssDNA in panels d-d’ is visualized with FAM_F’ ssDNA probe and fibronectin is visualized with a rabbit anti-fibronectin primary antibody and Alexa 647 secondary antibodies. See also: figure S1-S2 and movie S1 .

    Article Snippet: Hydrogels were then partially dried and a plastic clip-on 8-well slide with a silicone gasket (CCS-8, MatTek) was assembled around the region containing photopatterned ssDNA features and wells were incubated in 10-20 μg ml-1 bovine serum fibronectin (1 mg ml-1, F1141, Sigma) diluted in sterile 50 mM HEPES (pH 8.5) overnight at 4°C.

    Techniques: Labeling, Sequencing, Fluorescence, Staining

    ssDNA multiplexing supports production and long-term adhesion of patterned tissues. a . Schematic of iterative ssDNA photopatterning. b . Schematic for lipid-ssDNA labeling and patterning of multiple cell populations. Note surface fibronectin functionalization is not shown schematically, but is performed before cell capture. c . Confocal micrograph of polyT 20 F and polyT 20 G sequences photopatterned into a chess board pattern of alternating squares (250 μm side length) across an entire culture well. ssDNAs were sequentially patterned onto a 7.5%/0.25% (Am/Bis) BP-PA hydrogel by 254 nm UV, t = 60 s exposure for each ssDNA. Hydrogels were also functionalized with 20 μg ml -1 fibronectin. ssDNA patterns are visualized with 1 μM FAM_G’ and 1μM Cy5_F’ fluorescent ssDNA probes. d . Two populations of MDCKs expressing fluorescent H2B constructs captured on a similar chess board pattern. Cells were imaged t = 1 hr after capture and at t = 24 hrs. MDCK H2B-Venus cells were patterned using the G/G’ ssDNA pair and MDCK H2B-iRFP cells were patterned using the F/F’ ssDNA pair ( table S1 ). Inset , detailed view of interface. Data are representative of two independent experiments. See also: figure S3 .

    Journal: bioRxiv

    Article Title: Independent control over cell patterning and adhesion on hydrogel substrates for tissue interface mechanobiology

    doi: 10.1101/2022.11.16.516785

    Figure Lengend Snippet: ssDNA multiplexing supports production and long-term adhesion of patterned tissues. a . Schematic of iterative ssDNA photopatterning. b . Schematic for lipid-ssDNA labeling and patterning of multiple cell populations. Note surface fibronectin functionalization is not shown schematically, but is performed before cell capture. c . Confocal micrograph of polyT 20 F and polyT 20 G sequences photopatterned into a chess board pattern of alternating squares (250 μm side length) across an entire culture well. ssDNAs were sequentially patterned onto a 7.5%/0.25% (Am/Bis) BP-PA hydrogel by 254 nm UV, t = 60 s exposure for each ssDNA. Hydrogels were also functionalized with 20 μg ml -1 fibronectin. ssDNA patterns are visualized with 1 μM FAM_G’ and 1μM Cy5_F’ fluorescent ssDNA probes. d . Two populations of MDCKs expressing fluorescent H2B constructs captured on a similar chess board pattern. Cells were imaged t = 1 hr after capture and at t = 24 hrs. MDCK H2B-Venus cells were patterned using the G/G’ ssDNA pair and MDCK H2B-iRFP cells were patterned using the F/F’ ssDNA pair ( table S1 ). Inset , detailed view of interface. Data are representative of two independent experiments. See also: figure S3 .

    Article Snippet: Hydrogels were then partially dried and a plastic clip-on 8-well slide with a silicone gasket (CCS-8, MatTek) was assembled around the region containing photopatterned ssDNA features and wells were incubated in 10-20 μg ml-1 bovine serum fibronectin (1 mg ml-1, F1141, Sigma) diluted in sterile 50 mM HEPES (pH 8.5) overnight at 4°C.

    Techniques: Multiplexing, Labeling, Capture-C, Expressing, Construct