Tissuequest analysis software

In vitro blood-brain barrier (BBB) transmigration assays were performed as previously described^{29 (link)}. The artificial BBB was formed with either HUVECs or HBMECs (20,000 cells / well) in co-culture with HA cells (100,000 cells / well) for 3 days on Transwell-inserts with 3 μm fluoroblok membranes. Cell-tracker green (CMFDA)-labeled Br-M Ctrl or Br-M CTSS KD cells (20,000 cells / well) were allowed to transmigrate for 18h through the artificial BBB towards a FBS gradient, in the presence or absence of VBY-999 (10 μM). Tumor cell transmigration through empty inserts (coated with gelatin and poly-L-lysine) or inserts coated with HUVECs, HBMECs or HAs alone were used to determine the baseline migratory potential and the contribution of the single cell types to BBB formation. The number of transmigrated tumor cells was quantified by analyzing 200 fields of views (FOVs) that were acquired with a 20× objective (200× total magnification) using TissueQuest analysis software (TissueGnostics). Analysis was performed blinded to the group allocation.

For the analysis, fluorescently labeled slides were scanned using the TissueFAXS slide scanning system (TissueGnostics, GmbH, Vienna, Austria) based on a Zeiss Axio Imager Z2 upright epifluorescence microscope. Images were captured using a Zeiss 20X Plan-Apochromat air objective (0.8NA). Analysis was performed using TissueQuest analysis software (TissueGnostics, Vienna, Austria) that identifies cells based on segmentation of DAPI-stained nuclei based on thresholds set for intensity to identify all DAPI nuclei. From that, analysis parameters were set to identify immunostaining for PD-1, PD-L1, CD4, CD8, Foxp3, CD68, and Ki67 on a per cell basis utilizing the nuclei as a starting point. Immunostaining intensity was determined for each cell from the average pixel intensity for all pixels across the cell area based on an 8-bit grey scale. From the nuclei, the analysis software was programmed to look at a certain specified distance of 10 μm radially outward through the cell cytoplasm for immunostaining intensity above a threshold determined for specificity based on non-specific control samples. Measures of cell counts, mean intensity, percentage of positive cells out of the total number of cells (% positive cells/all nucleated cells) and positive cell density were determined using the TissueQuest analysis platform.

Cell counts were performed using the TissueQuest analysis software (TissueGnostics) on fluorescence images.

Fluorescent immunohistology was either performed on histological sections of TB-infected lung tissues that were either supplied by Dr. Pratista Ramdial of IALCH or prepared in-house from formalin-fixed lung tissue following resections. Sections were dried overnight at 60°C and then processed using an Opal 4-colour Manual IHC kit (Perkin Elmer) as per manufacturer’s instructions with CD20 (1:400), CD3 (1:400) and CD127 (1:100), VIP (1:100) and OSM (1:100) as primary antibodies. Slides were scanned on a Zeiss Axio Observer microscope using TissueFAXS imaging software (Tissuegnostics) and analysed using TissueQuest analysis software (Tissuegnostics).