Journal: Lab on a Chip

Article Title: Analysis of fast protein phosphorylation kinetics in single cells on a microfluidic chip

doi: 10.1039/C4LC00797B

Figure Lengend Snippet: Fig. 4 Time trajectories of single-cell PLA dot count distributions for the evaluation of protein phosphorylation kinetics. Left columns show the phosphorylation kinetics of the PDGF receptor and three kinases within the Akt pathway upon PDGF stimulation, and right columns rep- resent the phosphorylation kinetics of the IGF-1 receptor and three kinases within the Akt pathway upon IGF-1 stimulation. Each time point shown in the graphs represents the normalized PLA dot count distribu- tions (blue, in y-direction) evaluated from more than 400 single cells. Mean values are indicated in red.

Article Snippet: After stimulation, the cells were fixed with 4% formaldehyde (ThermoFisher Scientific) for 16 min at room temperature and permeabilized with 0.05% Tween 20 (Sigma Aldrich) for 3 min. On-chip proximity ligation assay Fixed cells were blocked (Olink) on-chip for 1 h. Next, monoclonal primary antibodies (Cell Signaling Technology) were diluted with antibody diluent (Olink) and incubated with the cell samples for 12 h with refresh cycles repeated every 2 h. The dilution ratio was 1 : 50 for Akt (Ser-473) (Cell Signaling, 4060), Akt (Thr-308) (Cell Signaling, 2965), and GSK-3β (Ser-9) (Cell Signaling, 9323), 1 : 25 for the PDGF β receptor (Tyr-751) (Cell Signaling, 3161), and 1 : 250 for the IGF-1 receptor (Tyr-1161) (Santa Cruz Biotechnology).

Techniques: Phospho-proteomics

Journal: Lab on a Chip

Article Title: Analysis of fast protein phosphorylation kinetics in single cells on a microfluidic chip

doi: 10.1039/C4LC00797B

Figure Lengend Snippet: Fig. 5 Phosphorylation rates evaluated from the time trajectories of single-cell PLA dot count distributions. The left and right columns represent the first 240 seconds of the protein phosphorylation time trajectories upon PDGF and IGF-1 stimulations, respectively. PLA dot counts of single cells are shown in gray. The mean values of the PLA dot counts are indicated by black dots. The simulated phosphorylation dynamics are shown in red.

Article Snippet: After stimulation, the cells were fixed with 4% formaldehyde (ThermoFisher Scientific) for 16 min at room temperature and permeabilized with 0.05% Tween 20 (Sigma Aldrich) for 3 min. On-chip proximity ligation assay Fixed cells were blocked (Olink) on-chip for 1 h. Next, monoclonal primary antibodies (Cell Signaling Technology) were diluted with antibody diluent (Olink) and incubated with the cell samples for 12 h with refresh cycles repeated every 2 h. The dilution ratio was 1 : 50 for Akt (Ser-473) (Cell Signaling, 4060), Akt (Thr-308) (Cell Signaling, 2965), and GSK-3β (Ser-9) (Cell Signaling, 9323), 1 : 25 for the PDGF β receptor (Tyr-751) (Cell Signaling, 3161), and 1 : 250 for the IGF-1 receptor (Tyr-1161) (Santa Cruz Biotechnology).

Techniques: Phospho-proteomics

Journal: Advanced Science

Article Title: Combinatorial Microgels for 3D ECM Screening and Heterogeneous Microenvironmental Culture of Primary Human Hepatic Stellate Cells

doi: 10.1002/advs.202303128

Figure Lengend Snippet: a) Representative maximum intensity projection of confocal images of HSCs cultured with 8C4 and 8FN scaffolds, the lowest and highest conditions for PDGFRβ expression. Light blue: DAPI. Purple: DiD membrane stain. Green: anti‐PDGFRβ. Scale bar: 100 µm. b) Box and whisker plots of the anti‐PDGFRβ fluorescence intensity per nucleus normalized to the average of all conditions per experimental replicate. n = 9 from three experimental replicates. c) Representative maximum intensity projection of confocal images of HSCs cultured with 8C1 and 8C4 scaffolds, the lowest and highest conditions for collagen I expression. Light blue: DAPI. Purple: DiD membrane stain. Yellow: anti‐collagen I. Scale bar: 100 µm. d) Box and whisker plots of the anticollagen I fluorescence intensity per nucleus normalized to the average of all conditions per experimental replicate. n = 9 from three experimental replicates. Two‐way interaction ANOVA. * p < 0.05 ** p < 0.01 *** p < 0.001 where * means between conditions and ^ means versus eight‐arm 8% counterpart.

Article Snippet: Microwells had their media removed and were fixed with 4% paraformaldehyde (RT15710, Electron Microscopy Sciences) in PBS for 20 min. Microwells were then washed twice with PBS and permeabilized with 0.5% Triton X‐100 (X100, MilliporeSigma) in PBS for 15 min. Microwells were washed once with PBS and blocked with 1% w/v bovine serum albumin (BSA, A2153, MiliporeSigma) in 0.1% Triton X‐100 in PBS for 1 h. Microwells were washed once with PBS and stained for 24 h with 2 μg mL −1 of anti‐human procollagen I alpha one antibody (AF6220, R&D Systems) and 0.848 μg mL −1 anti‐LOX antibody (ab174316, abcam) or 4 μg mL −1 anti‐human PDGFR beta antibody (AF385, R&D Systems) and 10 μg mL −1 anti‐human/mouse/rat alpha‐smooth muscle actin antibody (MAB1420, abcam) in 0.1% BSA and 0.1% Triton X‐100 in PBS for 24 h in a gentle shaker.

Techniques: Cell Culture, Expressing, Membrane, Staining, Whisker Assay, Fluorescence