Gen5 image software

Antifungal activity of CCL2 proteins against Botrytis cinerea strain BMM was tested in 96-well Costar cell culture plates (Corning Incorporated, Corning, NY, United States) in a total volume of 200 μL. Spores of B. cinerea were diluted in 25% PDB medium (Potato Dextrose Broth, Oxoid, Hampshire, United Kingdom) and used at a final density of 1 × 10³ spores mL^–1. Purified CCL2 proteins in 20 mM Na phosphate buffer pH 6 were added at final concentrations of 0–1000 μg mL^–1. After incubation on a shaking platform (80 rpm; 20°C), OD₅₉₅ was measured using the cell imaging multi-mode plate reader Cytation^TM 5 (BioTek, Winooski, VT, United States). The absorbance reads were analyzed with Gen5 Image + Software (Version 3.03.14, BioTek). Growth curves were generated by GraphPad Prism version 8.0.2 (GraphPad Software, Inc., La Jolla, CA, United States). Experiments were repeated 3-times.

Adult ASM cells were plated in a 12 well plate at a density of 8000 cells per well in DMEMF12 containing 1% FBS and 1% Antibiotic/Antimycotic. Cells were allowed to grow for 3 days, fixed with 4% paraformaldehyde, permeabilized, immunostained for Ki67 (Abcam ab9260, 1:200) using standard procedures (nuclei stained with DAPI) and imaged using fluorescence microscopy [30 (link)]. Positive cells were determined in each sample from 25 randomly selected fields at 10x magnification using a Bio-Tek Cytation5, analyzed using a visual threshold by Bio-Tek Gen5Image software, and expressed as percentage of total cell number (DAPI counterstain). All assays were done in triplicate, and at least 200 cells were counted.

All mice were sacrificed 24 h after CLP and blood collected via cardiac puncture. Samples were centrifuged for 10 min at 4°C with 1000 g. Serum was procured from each sample for analysis. Soluble cytokine concentrations of IL-1β, IL-6, IL-10, MCP-1, TNF-α, and INF-γ were determined using the Bio-Plex 200 System with suspension array kits according to the manufacturer’s instructions (Bio-Rad). All samples were run in duplicate. Results were analyzed using the Bio-Plex Manager 3.0 Software. Levels were reported in pg/mL. Organ damage marker concentrations were obtained from serum by ELISA using the BioTek Synergy HT plate reader (BioTek). These markers included ALT (ABclonal), AST (Abcam), and creatinine (Arbor Assays). All kits were used according to the respective manufacturer’s instructions. Results were analyzed using Gen5 Image+ software (BioTek). Levels were reported in pg/dL and mg/dL.

HAEC were plated and allowed to grow to confluent monolayers and then treated according to the protocol, as mentioned above. Monocytes (SC cells) were labeled with 8 μM CellTracker Green (CMFDA; Invitrogen, Eugene, OR, USA) and then treated following the protocol used for the HAEC. After treatment, 1 × 10⁶ monocytes were added to the endothelial monolayers and incubated at 37 °C for 45 min. The non-adherent cells were washed away with EC media and collected. Phase-contrast images of HAEC and monocytic cells were created using a Cytation 5 Cell Imaging Multi-Mode Reader with a 10× microscope objective and then merged. The merged images were used to quantify Celltracker Green labeled monocytes in the green channel. In each image, the total green signal associated with the level of monocyte adherence was quantified using Gen5 Image+ software (BioTek, Winooski, VT, USA). In addition, both adherent cells and non-adherent cells were lysed in 0.2% Triton X for quantification. The fluorescent intensity of the monocytes added to the monolayer (input), as well as that of the non-adherent cells, was measured at an excitation of 485 nm and an emission of 528 nm. Results are expressed as a percentage of control.