Gen5 imaging software

Yeast strains were inoculated in 3 mL of non-inducing media and allowed to grow overnight at 30 °C. The following evening, cultures were spun and washed twice in water, then resuspended in inducing growth media and returned to 30 °C for overnight growth. Following a minimum of 16 h induction, yeast cells were pipetted onto glass slides and sealed under coverslips using nail-polish. Fluorescence microscopy was performed using the Cytation5 cell imaging multi-mode reader (BioTek, Winooski, WI, USA). The following LED cubes and imaging filter cubes from biotech were employed for GFP tagged proteins: 465 LED 1225001 Rev J, GFP 469/525 1225101 Rev J, BioTek; and for CFP-tagged proteins: 465 LED 1225001 Rev J, CFP 445/510 1225107 Rev I. Images were captured on the 20× objective. Image analysis was completed using the Gen5 imaging software (BioTek).
Yeast microscopy data were quantified by taking three biological fields on the 20× objective and overlaying a grid to cut the image into four quadrants. Total of 50 cells were counted and sorted as either showing a diffuse signal or containing one or more fluorescent foci for each biological field. Stacked bar graphs with SD error bars were generated in GraphPad Prism using this data.

L929 cells in all culture wells were examined daily during the experiment under a light microscope (Olympus CKX41, Olympus Europa SE & Co. KG, Hamburg, Germany). Microscopic imaging of representative wells was performed using a Cytation 5 Cell Imaging Multi‐Mode Reader (BioTek Instruments GmbH, Bad Friedrichshall, Germany). Imaging was performed in brightfield using the intrinsic auto‐exposure function of the Gen5 imaging software (Version 3.10.06, BioTek Instruments GmbH, Bad Friedrichshall, Germany) for 4x or 20x objectives.

In order to determine the cell viability after 28 days of culture, the constructs were first washed in Dulbecco’s phosphate buffered saline (DPBS, Gibco) before being stained with a Live/Dead staining kit (ThermoScientific) according to the manufacturer’s protocol. Images were acquired on a Cytation 5 multi-mode reader using Gen5 imaging software (BioTek, Winooski, VT, USA). The area of positive calcein stain was determined in two fields per sample using a custom MATLAB code utilizing MATLAB R2020a software (Figures S1 and S2).

After sacrifice, the dorsal skin samples were fixed immediately in 4% paraformaldehyde and embedded in paraffin. Sections were then cut and stained with hematoxylin and eosin (H&E), Congo red, or toluidine blue. The number of eosinophils and mast cells was counted in three randomly selected areas per sample at x200 using a Lionheart FX microscope equipped with Gen5 imaging software (BioTek Instruments, Winooski, VT, USA).