polyacrylamide gel substrates  (Bio-Rad)

Journal: Scientific reports

Article Title: Mechanosensitive traction force generation is regulated by the neutrophil activation state.

doi: 10.1038/s41598-023-37997-y

Figure Lengend Snippet: Figure 1. Neutrophils from septic patients produce greater forces and traction maxima than neutrophils from healthy donors. Traction force microscopy of unstimulated neutrophils from either a healthy donor (n = 293) or septic donor (n = 24) on 1.5 kPa polyacrylamide gel substrates, quantifying mean and standard error mean (SEM) of (a) root-mean-square displacement ( uRMS ), (b) total force ( F ), (c) root-mean-square traction ( TRMS ), and (d) traction maxima ( TMax ). Cells were maintained at 37 ◦C throughout the duration of the experiment. Displacements were computed in Matlab by T-PT, and displacement heatmaps |u| (e) and traction heatmaps |t| (f) were generated for two representative cells at 30 min; forces and tractions were computed using the finite element method in Abaqus. Scale bars are 10 μm, and the white contours represents the boundary of the cell edge. ***p < 0.001, ****p < 0.0001 (unpaired Student’s t-test).

Article Snippet: Polyacrylamide gel substrates were then prepared using varying concentrations of acrylamide (Bio-Rad) and N,N-methylene-bisacrylamide (Bio-Rad) to achieve either a Young’s modulus of either 1.5 kPa or 10 kPa; 1.5 kPa substrates were made with 3% acrylamide and 0.2% bisacrylamide, and 10 kPa substrates with 5.2% acrylamide and 0.19% bisacrylamide.

Techniques: Microscopy, Generated

Journal: Scientific reports

Article Title: Mechanosensitive traction force generation is regulated by the neutrophil activation state.

doi: 10.1038/s41598-023-37997-y

Figure Lengend Snippet: Figure 6. PMA does not increase traction force generation on stiff substrates. Mean (and SEM) of RMS tractions ( TRMS ), traction maxima ( TMax ), and displacements of neutrophils from healthy donors seeded on polyacrylamide gels with a Young’s modulus of either 1.5 kPa (n = 70) (a–c) or 10 kPa (n = 105) (d–f). Cells were allowed to attach and settle, and then imaged before stimulation (“Unstim.”). Cells were then treated with 20nM PMA and were imaged 5 min post-addition and 30 min post-addition of PMA. Tractions were computed using the finite element method in Abaqus, and displacement heatmaps were generated for two representative cells at 30 min (c,f). Scale bars are 10 μm, and the white contours represents the boundary of the cell edge. Analyzed using paired Student’s t-test comparing each 5 min timepoint to the unstimulated timepoint and comparing each 30 min timepoint to the unstimulated timepoint, *p < 0.05.

Article Snippet: Polyacrylamide gel substrates were then prepared using varying concentrations of acrylamide (Bio-Rad) and N,N-methylene-bisacrylamide (Bio-Rad) to achieve either a Young’s modulus of either 1.5 kPa or 10 kPa; 1.5 kPa substrates were made with 3% acrylamide and 0.2% bisacrylamide, and 10 kPa substrates with 5.2% acrylamide and 0.19% bisacrylamide.

Techniques: Generated

Journal: Scientific reports

Article Title: Mechanosensitive traction force generation is regulated by the neutrophil activation state.

doi: 10.1038/s41598-023-37997-y

Figure Lengend Snippet: Figure 10. β-glucan priming increases RMS traction and traction maxima during fMLF stimulation on both soft and stiff substrates. Mean (and SEM) of RMS tractions ( TRMS ) and traction maxima ( TMax ) of neutrophils from healthy donors seeded on polyacrylamide gels with a Young’s modulus of either 1.5 kPa (soft) or 10 kPa (stiff). Cells were allowed to attach and settle and imaged before the addition of β-glucan or fMLF (“Unstim.”). (a–d) Cells with “No Priming” were then treated with 1 μM of fMLF and imaged 5 min and 30 min post- addition; the RMS tractions TRMS on 1.5 kPa (a) and 10 kPa (b) substrates and the traction maxima TMax on 1.5 kPa (c) and 10 kPa (d) substrates were calculated using the finite element method in Abaqus (1.5 kPa, n = 27; 10 kPa, n = 107). (e–h) Cells with “Priming with β-glucan” were first primed with 20 μg/mL soluble β-glucan for 10 min and then treated with 1 μM of fMLF and imaged 5 min and 30 min post-fMLF addition; the RMS tractions TRMS on 1.5 kPa (e) and 10 kPa (f) substrates and the traction maxima TMax on 1.5 kPa (g) and 10 kPa (h) substrates were computed using the finite element method in Abaqus (1.5 kPa, n = 68; 10 kPa, n = 48). Analyzed using paired Student’s t-test comparing each 5 min timepoint to the unstimulated timepoint and comparing each 30 min timepoint to the unstimulated timepoint, *p < 0.05.

Article Snippet: Polyacrylamide gel substrates were then prepared using varying concentrations of acrylamide (Bio-Rad) and N,N-methylene-bisacrylamide (Bio-Rad) to achieve either a Young’s modulus of either 1.5 kPa or 10 kPa; 1.5 kPa substrates were made with 3% acrylamide and 0.2% bisacrylamide, and 10 kPa substrates with 5.2% acrylamide and 0.19% bisacrylamide.

Techniques: