Relative levels of biofilm development were assessed using flow cells (Kharadi & Sundin, 2019 (link)). Strains expressing GFP via vector pMP2444 (Table 1 ) were grown overnight and normalized at an OD600 of 0.5. Flow cell chambers in a µ‐Slide VI (Ibidi) were inoculated with the cultures and incubated for 1 h at 24°C. Following this, a flow of 0.5 × LB medium was applied to the flow chambers using a peristaltic pump (Ismatec REGLO; Cole‐Parmer) for 5 h. Biofilms were visualized using a FluoView 1000 confocal laser‐scanning microscope (Olympus). z‐Stacked images of the flow cell channels were processed by ImageJ and compared for the green fluorescence value using the RBG assessment plug‐in (Schneider et al., 2012 (link)). Three replicates conducted in the study were statistically compared using Tukey's HSD on JMP statistical software.
Slide 6
Manufactured by Ibidi
The µ-slide VI is a specialized laboratory equipment designed for various cell culture applications. It features a grid-patterned surface to facilitate the cultivation and observation of cells. The µ-slide VI provides a controlled environment for maintaining and studying cellular samples.
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4 protocols using slide 6
1
Quantifying Biofilm Development via Flow Cells
2
Real-time imaging of RBC calcium dynamics
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RBCs were washed twice in RPMI and labelled for 30 min with 5 µM Fluo4-AM at 37 °C. After three washes with RPMI cells were re-suspended in complete medium and allowed to de-esterify at room temperature for 30 min. During this time cells were allowed to settle in poly-L lysine (PLL) coated slide chambers (Ibidi µ-slide VI). Non-adherent cells were removed by repeated washes. Alternate cycles of acquisitions every 4 s of brightfield and fluorescent signals were started before the addition of complete medium containing 5 µM Yoda1, 1 mM Jedi2 or DMSO vehicle and followed over about 5 min. Using Fiji software [63 (link)] relative fluorescent signals for defined areas of identical size covering individual RBCs were obtained. Background fluorescence was obtained by calculating the mean of five identical areas without cells and subtracted from all values.
3
Neutrophil Motility under Shear Stress
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To prepare flow chambers, Ibidi µ-Slide VI0.1 were coated with 0.5 μg/mL E-selectin, 7 μg/mL ICAM-1, and 8 μg/mL CXCL1 for 2 hours in PBS and then blocked with an excess of casein for 2 hours, both at room temperature. Flow chambers were perfused at 12.98 μL/min, which is calculated to produce a shear stress of 1 dyne/cm2. CFSE-labeled wild-type and vehicle control-treated vinculin knockout neutrophils were evaluated within the same flow chamber. Time-lapse images were captured every 10 seconds for 1 hour, starting immediately after starting flow chamber perfusion, using transmitted light through a 20X objective. Motility was tracked using ImageJ Manual Tracking and analyzed using Ibidi Chemotaxis to obtain measures of migration: accumulated distance, Euclidean distance, and velocity.
4
Coculture of Bacillus subtilis Strains
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We loaded cocultures of a PcomX-gfp ΔcomS mutant (responders) with the comS-overexpressing strain 184comS PcomX-rfp (senders) into two commercial microfluidic slides (IBIDI µ-slide VI) using static (not flowing) FMC medium and various ratios of comS overproducers/mutant ΔcomS responders (percentage by volume of cultures at an OD600 of 0.1 subjected to vortex mixing together). Strains PcomX-rfp, PcomX-gfp ΔcomS, PcomX-rfp plus 50 nM XIP, and PcomX-gfp ΔcomS plus 50 nM XIP were used as controls. The end ports of the channels were sealed with mineral oil to prevent drying of the medium in the channels. Images were taken as described for the microfluidic experiments, and analysis was performed similarly. In the case of the controls, XIP was added to planktonic culture and the tube subjected to vortex mixing before pipetting into the slide. Because the population was heterogeneous with respect to both fluorescent reporter type and comX expression, a fluorescence threshold was defined as the maximum RFP fluorescence observed in the PcomX-rfp negative control as described previously. The median of the RFP fluorescence observed above this cutoff level in other samples was used as a measure of how strongly the red cells were activating comX as a function of their number density.
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