Spectroflo software

To assess T cell subset frequencies, PBMCs were analyzed using a 25-color T cell phenotyping flow cytometry panel (Supplementary Table 3), using a standardized method previously published^{47 (link)}. Cells were analyzed on a five-laser Cytek Aurora spectral flow cytometer. Spectral unmixing was calculated with prerecorded reference controls using Cytek SpectroFlo software (v2.0.2). Cell types were quantified by traditional bivariate gating analysis performed with FlowJo cytometry software (v10.8).

Bronchoalveolar lavage fluid was collected from the lungs of P.a. infected and uninfected control mice 48 h.p.i. Mice were euthanized and 20G catheter was inserted into the trachea of mice. Then the BALF was collected by washing (3X) the lungs of infected and control mice with 0.5 ml PBS. Then the BALF was centrifuged at 500g for 10min, the supernatant collected for ELISA, and the cells pellets resuspended in PBS for flow cytometry. For flow cytometry, the cells were enumerated using an automatic cell counter (NanoEnTek), incubated with mouse FcBlock (BioRad) for 15 minutes at room temperature, then washed with PBS. Then cells were immunostained with a mixture of flurochrome-conjugated antibodies (α-CD45, α-CD11b. α-Ly6C, α-Ly6G, α-F4/80, and α-CCR2) for 30 min, at 4°C in the dark. After staining, cells were washed and resuspended in buffer for analysis. Flowcytometry data acquisition was performed on Cytek Aurora using SpectroFlo software (Cytek Biosciences, CA, USA) and data analyses were performed using FlowJo Software (FlowJo LCC, OR, USA).

EV-coated beads or CTV-labeled EVs were analyzed on a Cytek Aurora spectral cytometer within the Michigan State University Flow Cytometry Core Facility. Prior to each acquisition of CTV-labeled EVs, a Long Clean cycle was performed on the instrument, and Milli-Q water was run for a minimum of 30 min on high. CTV-labeled EVs were detected by utilizing a violet laser (405 nm) threshold (V3 detector; 451–466 nm) to coincide with CTV excitation/emission (405/450 nm). Threshold settings were determined based on PBS and PBS + CTV + FITC-annexin A5 staining reagents without EV controls. CTV-labeled EV samples were acquired for 10 s each to normalize acquisition time for all samples and controls. Signals from FITC and APC were unmixed using the SpectroFlo software (v3.0.1; Cytek Biosciences, California, CA, USA) using autofluorescence extraction and analyzed using FCS Express (v7; De Novo Software). Due to fluorescence thresholding settings, single beads were gated for the bead-based assays, while all events were analyzed in the CTV-labeled EV assays. Fluorescence minus 1 (FMO) controls were used as gating controls for both EV-coated bead and CTV-labeled EV assays with FITC (annexin A5 or GlaS) and APC (anti-CD63 or anti-annexin A1 antibodies) labeling.

Cells were synchronized by serum starvation for 72 h prior to treatment with 5 or 10 nM paclitaxel, 0.75 µM SMI#9 or both for 24 or 48 h. Cells were fixed with 70% ethanol, incubated with DNase free RNase A, stained with propidium iodide (PI) and measured using a Northern Lights full spectrum flow cytometer (3-laser configuration; Cytek Biosciences, Fremont, CA) at the Microscopy, Imaging, and Cytometry Resources Core of Karmanos Cancer Institute. An unstained control was used to unmix cellular autofluorescence from PI fluorescence during acquisition using SpectroFlo software (Cytek). Flow Cytometry Standard (FCS) files were exported, then imported into FlowJo (FlowJo LLC, Ashland, OR) where they were gated to remove cell aggregates (based on PI-Height vs PI-Area) and the 2 N DNA peak position was normalized. Gated, normalized FCS files were exported, then imported into ModFit LT v.5.0 (Verity Software House, Topsham, ME) which deconvoluted the DNA histograms using the tetraploid model with debris and apoptosis modeling.

To perform the cell cycle study, fixed gem cells were stained for 1 h at 4°C with 6.5 μg/ml Hoestch 33342/PBS solution and were analysed in a Cytek Aurora flow cytometer as previously described (Bastos et al., 2005 (link)). Three peaks with increasing Hoechst fluorescence could be detected as representative of haploid (1C), diploid (2C) and tetraploid (4C) cells. We used a volumetric measurement during sample acquisition, using a volume sensor, that allows the calculation of events per µl for any given population and thus determine the absolute number of cells in the analysed sample. The data have been acquired and analysed using the SpectroFlo software (Cytek Biosciences).

Mouse spleen cells were washed with PBS with 1% BSA (staining buffer), blocked with mouse IgG in staining buffer, then stained with conjugated antibodies. Antibodies used were anti-CD11b (clone M1/70, Biolegend, San Diego, CA), anti-Ly-6C (clone HK1.4, Biolegend), anti-Ly-6G (clone 1A8, Biolegend), anti-F4/80 (clone BM8, eBiosciences, San Diego, CA), anti-MHCII (clone M5/114.15.2, Biolegend), anti-CD4 (clone RM4-5, Biolegend), anti-PD-1 (clone 29F.1A12, Biolegend), anti-Foxp3 (clone FJK-16s, eBiosciences), anti-Tbet (clone 4B10, Biolegend), and anti-RORγt (clone AFKJS-9, Biolegend). Prior to anti-Foxp3, anti-Tbet, and anti-RORγt staining, the cells were fixed and permeabilized.
Stained cells were analyzed in the Oklahoma Medical Research Facility (OMRF) Flow Cytometry Core Facility on a BD LSRII (BD Biosciences) or the DMEI Ocular Immunobiology Core on a 4-laser Aurora (Cytek Biosciences, Fremont, CA). When the Aurora was used, unmixing was done using SpectroFlo Software (Cytek) and data was analyzed using FlowJo Software (Tree Star, Inc., Ashland, OR).