Celltracker blue cmac

The parasite clumping assay was done as described previously (20 (link)), with modifications indicated below. A total of 5 × 10⁵ of CellTracker blue CMAC (Thermo Fisher Scientific)-prelabeled T. vaginalis organisms were plated at 1 × 10⁶/ml on glass coverslips covered with confluent Ects or coverslips alone and incubated in complete keratinocyte-SFM (serum-free media) with no CaCl₂ or 1 mM CaCl₂ for 30 min. Parasites were then fixed in 4% formaldehyde in PBS and mounted on slides using Mowiol (Calbiochem). Fifteen images of each coverslip were acquired using an Axioscope 2 epifluorescence microscope (Zeiss) and analyzed using Zen lite software. A clump is defined as an aggregate of 10 or more parasites. To calculate clumping fold changes, EV − Ca + host was set as 1-fold, so CLP − Ca would be 5.95-fold and EV − Ca would be 0.025-fold; thus, CLP − Ca/EV − Ca = a 238-fold increase. CLP − Ca + host is 7.47-fold and EV − Ca + host is 1-fold; thus, CLP − Ca + host/EV − Ca + host = a 7.47-fold increase.

Attachment of T. vaginalis to Ects was performed as described previously (10 (link)). Briefly, 5 × 10⁴ CellTracker blue CMAC (7-amino-4-chloromethylcoumarin; Thermo Fisher Scientific)-labeled T. vaginalis organisms were incubated with confluent Ects for 30 min, and the coverslips were fixed in 4% formaldehyde in PBS and mounted on slides using Mowiol (Calbiochem). Fifteen images of each coverslip were acquired using an Axioscope 2 epifluorescence microscope (Zeiss), and cell counts were quantified using Zen lite (Zeiss) and ImageJ software (74 (link)).
Attachment assays that included rCLP ECD to compete for Ect binding were performed using WT, nontransfected RU393 parasites, and the difference in the procedure was addition of 0.25 μg, 1 μg, or 4 μg of rCLP ECD or 4 μg of BSA as a negative control to Ects for 30 min prior to parasite addition. Medium was then removed and replenished with new medium containing CellTracker blue-labeled parasites.

Photoconversion studies were done on mosaic populations of MDCK-GFP-HRas^V12 cells co-cultured (on soft or stiff ECM) with MDCK cells that had been transiently transfected with mEos2-FilaminA-N-9. To distinguish mEos2-Filamin-expressing green normal cells from GFP-HRas^V12-expressing green transformed cells, we stained the former with CellTracker Blue CMAC (Thermofisher), according to manufacturer protocol, before creating the mosaic monolayer. An optimally expressing mEos2-Filamin cell was chosen that interfaced with an MDCK-GFP-HRas^V12 cell. Stimulation was done on a point region-of-interest in the mEos2-filamin cell using a 405 nm laser at 2% intensity, looped over for 25 times with a scan speed of 1000 μs/pixel. This was immediately followed by LSM imaging of the green and red channels; 0.3% intensity, 700 V PMT voltage for the green and 4% intensity, 650 V PMT voltage for the red channels and filamin dynamics was subsequently tracked for 180 s.
Photobleaching studies were done using MDCK cells stably expressing mApple-FilaminA, cultured on the soft or stiff substrate. 561 nm laser was used at 5% intensity for bleaching a region-of-interest, iterated or looped over five times with a scan speed of 200 μs/pixel. For LSM imaging, the laser power was attenuated to avoid phototoxicity. Images were collected before, immediately after, and for 60 s following the bleaching.