Shandon cytospin 4 centrifuge

Cells were spotted on slides using Shandon Cytospin4 centrifuge (Thermo Scientific), and fixed for 10 minutes at RT in 4% paraformaldehyde. For nuclear antigens, cells were incubated in Methanol for 5 minutes at -20°C prior to blocking. Samples were blocked for 30 min at RT in blocking buffer (1% BSA, 10% fetal goat serum, and 0.2% saponin) and incubated overnight at 4°C, or 1 hour at RT, with primary antibodies (S2 Table) diluted in blocking buffer. Slides were washed three times in TTBS, and incubated with the corresponding secondary antibody conjugated to Alexa fluorophores (1:1000, all from Invitrogen). DNA was stained with DAPI. The slides were mounted with Fluorescent Mounting Medium (GBI Labs). Images were visualized under a fluorescent BX61 microscope or TCS SP5 confocal fluorescent microscope (Leica). To demonstrate antibody specificity, a minus-primary antibody control was employed.

Wild type and C3^−/− mice (Jax Laboratories: B6.129S4-C3^tm1Crr/J) were bred in house according to the approval by University of Colorado Animal Protocol Committee. NWs were injected as a 5 mg/kg bolus via tail vein into WT and C3 KO mice (8 weeks of age, females). Following the injection (5 min), mice were sacrificed and the blood was drawn via cardiac puncture using heparin as anticoagulant. Blood was applied on Miltenyi Mini MACS magnetic column (Miltenyi Biotech) and the trapped cells and particles were washed extensively with PBS. The particles and magnetic leukocytes were then eluted from the column. The cells were pelleted with tabletop Eppendorf centrifuge and the particles in the supernatant were further concentrated with ultracentrifuge for C3 dot-blot assay. The cell pellet was resuspended in 200 μL PBS and the cells were concentrated on slides using Shandon Cytospin 4 centrifuge (Thermo Fischer), fixed with 10% (v/v) formalin in PBS, and stained with Hoechst dye (Thermo Fischer) to enable leukocyte nuclei visualization. The images were taken with Nikon Eclipse E600 fluorescent microscope using DAPI filter at low (40×) magnification.

Cell concentration for all 126 BAL1-BAL2 sample pairs were assessed using an automatic Vi-Cell XR Cell Viability Analyzer (Beckman Coulter, Mississauga, ON, Canada). For confirmation, a manual cell count using trypan blue dead cell exclusion on a hemocytometer was performed on nine randomly selected sample pairs with quantification of ciliated epithelial cells. Cytospin preparations of 6 of these sample pairs were made: 150uL of the whole (raw) BAL was loaded into a Cytospin filter attached to a charged glass slide. The Cytospin filters with glass slides were then centrifuged at 800 rpm for 3 min (Shandon Cytospin 4 Centrifuge, ThermoFisher). The slides were then air dried and stained with HARLECO® Hemacolor® (EMD Chemicals, NJ, USA). Percentages of polymorphonuclear granulocytes, eosinophils, macrophages, and lymphocytes were quantified. All manual sample analyses were performed by experienced operators blinded to sample group.

Cells were spotted on slides using Shandon Cytospin4 centrifuge (Thermo Scientific), and fixed for 15 minutes at room temperature in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA). Samples were blocked for 30 min at room temperature in blocking buffer [1% BSA (Sigma-Aldrich), 10% fetal goat serum (Biological Industries), and 0.2% saponin (Sigma-Aldrich)] and incubated overnight at 4 °C with primary antibodies (see Supplementary Table S5) diluted in blocking buffer. Slides were washed 5 times in PBS containing 0.1% Tween 20 (Sigma- Aldrich), and incubated for 40 min at room temperature with a secondary antibody conjugated to Alexa fluorophores (1:1000, Life Technologies). For rabbit anti-ARX, rabbit anti-PAX4, and mouse anti-PDX1 detection, samples were incubated with biotin-conjugated anti-rabbit/mouse IgG, respectively (1:200, Jackson ImmunoResearch, West Grove, PA) for 30 min at room temperature, followed by 40-min incubation at room temperature with Cy3-streptavidin (1:900, Jackson ImmunoResearch). The slides were mounted with Fluorescent Mounting Medium containing DAPI (GBI Labs, Bothell, WA). Images were taken using a Zeiss LTM 200 fluorescent microscope or a Leica SP5 confocal microscope. To demonstrate antibody specificity, a minus-primary antibody control was employed.

Cytospins were prepared on coated slides with 1 × 10⁵ cells using the Thermo Fisher Scientific Shandon Cytospin 4 centrifuge. The slides were then stained with May–Grünwald–Giemsa solution (Catalog No. MG500, Sigma Aldrich) for 5 min. After rinsing for 90 s in 40 mM Tris buffer at pH 7.4, the slides were stained with Giemsa solution (Catalog No. GS500, Sigma-Aldrich) for 15 min. Finally, the slides were rinsed twice with water. The cells were imaged using a Leica DM2000 inverted microscope.

The effect of VPE, AP, and H, alone or in combination with 5-FU, on HCT116 and RKO cell death (48 h of pre-incubation followed by 48 h of co-incubation), was evaluated by fluorescence microscopy assessing the presence of nuclear condensation, as previously described [26 (link)]. Briefly, both floating and attached cells were collected, fixed with 4% (w/v) paraformaldehyde, washed with PBS, and attached to poly-L-lysine coated slides using a Shandon Cytospin 4 centrifuge (Thermo Scientific, Waltham, MA, USA). Then, the slides were stained with Hoechst 33342, and analyzed by fluorescence microscopy. At least 500 randomly selected cells were counted per sample, and the percentage of apoptotic cells was calculated from the ratio between cells presenting nuclear condensation and the total number of cells.