Improved longevity of actomyosin in vitro motility assays for sustainable lab-on-a-chip applications

Figures

• 

  Fig. 1. Assembled conventional flow cells. (A) Schematic drawing of open non-sealed flow cells with access to fluid via left and right openings of each cell. (B) Schematic drawing for flow cell assembly after addition of silicone vacuum grease to seal the cells. (C) Photography of assembled flow cell (with roof coverslip indicated by dashed lines) with droplets of fluid outside flow cell openings to avoid evaporation of fluid from the flow cell itself.

• 

  Fig. 2. Time-dependent decrease of motility quality is associated with a drop in pH in open flow cells. (A) Relative sliding velocity in open flow cells (velocity in open cell/velocity in closed cell) vs. time after adding assay solution. Each data point represents mean ± 95% CI based on three independent experiments except point at 150 min, representing two experiments. The experiments were performed in a paired design (open vs. closed cell) on three different days with different stock solutions including myosin and actin batches and partly different personnel. Data for individual experiments in open and sealed flow cells are presented in Fig. S2. (B) Relative fraction of motile filaments (fraction in open cell/fraction in closed cell) vs. time as in (A). Each data point is given as mean ± 95% CI based on three or two independent experiments (same as in A). (C) pH vs. time for open (black) and sealed (orange) flow cells, estimated using pH paper (Figs. S3, S4). The solid lines represent linear regression curves fitted to the underlying data and given with 95% CI (dotted lines). pH was 7.4 at start of all three experiments. Data from one experiment at times 30–120 min (other flow cell…

• 

  Fig. 3. Loss of actin filaments during multiple exchanges of assay solution. Rhodamine phalloidin labeled actin filaments are shown in gray. The images are taken after each consecutive assay solution exchange at 20, 40 and 60 min, from left to right. A visibly lower concentration of actin filaments is seen with each solution exchange. Scale bar, 10 μm.

• 

  Fig. 4. Loss of actin filaments from surface to the fluid phase, compared to good motility. Summed maximum intensity projection (Z-projection in ImageJ) of 20 consecutive grayscale images of Rhodamine phalloidin labelled actin filaments captured at 5 FPS for 4 s. (A) A vast majority of the actin filaments were floating in solution and were not attached to the surface as indicated by blurred projection reflecting Brownian motion. (B) Attachment of all filaments with good motility quality as indicated by extended paths traced out by a majority of the actin filaments. Image intensities adjusted for clarity. Scale bar, 10 μm.

• 

  Fig. 5. Average gliding velocity and fraction of motile filaments vs. time for 6 different experiments. (A) Gliding velocity. (B) Fraction of motile filaments. a60: protocol using a60 assay solution with storage at 21 °C on wet paper between motility assays (performed at 25 °C) showing results of the 2 successful experiments. Individual data in Fig. S6. a45i: protocol using a45 assay solution without objective based temperature control, but storage of the flow cells on the microscope stage between in vitro motility assays, starting with room temperature (21 °C) but with temperature of ∼25 °C after 4 h. Individual data in Fig. S8. a45ii: protocol using a45 assay solution with storage of flow cell on the microscope stage between in vitro motility assays with temperature in the range 24–25 °C during the entire experiment. Individual data in Fig. S9. Note, the initial increase in velocity and fraction of motile filaments from 0–4 h in the a45i protocol can be attributed to the increase in temperature. Note further that both velocity and fraction of motile filaments in the a45i protocol reach approximately the same values as in the other protocols at 3–4 h. Note, finally, that whereas the…

• 

  Fig. 6. Gliding velocities and fraction of motile filaments vs. time using a microfluidic platform. (A) Gliding velocities vs. time after the addition of assay solution with the microfluidic chamber sealed (orange) or open (magenta). Each time point is represented by mean ± 95% CI based on measurements on 15–18 filaments. (B) Fraction of motile filaments vs. time in 3 regions of interest of the same flow cells as in A, either sealed (orange) or open (magenta). Each time point is represented by mean ± 95% CI. T = 24–25 °C.