Hoechst 33342 nuclear stain

Endothelial basal medium-2 (EBM-2) and Astrocyte Growth Medium (AGM) bullet kit were from Lonza Inc., Walkersville MD. Rat Plasma Fibronectin was from EMD Millipore, Billerica MA. TrypLE Select, Rat Tail Collagen Type I, Recovery Cell Culture Freezing Medium, Alexa Fluor^® 488 Phalloidin, 10% Normal Goat Serum, Texas Red-dextran (40 kDa), Hoechst 33342 nuclear stain were from Life Technologies Corporation, Carlsbad CA. Hyclone Phosphate Buffered Saline (PBS), tissue culture treated T75 culture flasks, BD Tuberculin Slip Tip 1ml syringe were from Fisher Scientific, Pittsburgh PA. 99.9% Methanol, Glacial Acetic Acid, 96% Paraformaldehyde and Draq5 Fluorescent Probe were from Thermofisher Scientific, Rockford IL. Premium Select FBS was from Atlanta Biologics, Lawrenceville GA. Bovine Plasma Derived Serum (BPDS) was from Animal Technologies Inc., Tyler TX. Tygon tubing (Cat. Number: AAD04103) was from Saint Gobbin PPL Corp., Valley Forge PA. 1000 μl gas tight syringes were from Hamilton Laboratory Products, Reno NV.

Cell surface staining was performed using a primary CD71 antibody (catalog no. PA5-27739; Sigma-Aldrich) or negative control antibody (rabbit immunoglobulin fraction, catalog no. X0936; Dako) followed by goat antirabbit Alexa Fluor 488 (catalog no. A-11008; Life Technologies) and counterstained with DAPI (catalog no. P36941; Life Technologies). To visually confirm/image phagocytosis, 1 × 10⁵ whole BAL cells were placed in an 8-well chamber slide (catalog no. 734-2050; VWR) in live cell imaging solution containing 1% bovine serum albumin (catalog no. A14291DJ; Life Technologies). Bacterial phagocytosis was captured 2 hours after addition of 100 μl of 0.25 mg/ml pHrodo Green S. aureus BioParticles (catalog no. P35382; Life Technologies) for 2 hours at 37°C, 5% CO₂, with Hoechst 33342 nuclear stain (catalog no. R37605; Life Technologies) and imaged using a Leica DM2500 microscope (Leica Microsystems), and data were analyzed using ImageJ-win32 software. Staining for iron in cytospin preparations was performed using a Prussian blue test kit (catalog no. HT20; Sigma-Aldrich).

Apoptosis was determined by Hoechst 33342 DNA fragmentation assay. Briefly, cells were incubated with 10 μg/ml Hoechst 33342 nuclear stain (Life Technologies, Carlsbad, CA, USA) for 30 min at 37 °C and percentage of cells having intensely condensed chromatin and/or fragmented nuclei by fluorescence microscopy (EVOS All-in-one digital inverted fluorescence microscope, Thermo Fisher Scientific, Waltham, MA, USA) were scored. From random fields, nuclei were analyzed for each sample. The apoptotic index was calculated as apoptotic nuclei/total nuclei x 100 (%) using ImageJ software (Java image processing, NIH, Bethesda, MD, USA).

MDA-MB-231 and 4T1 cells were co-transfected with mScarlet or mScarlet-C-SARAF and EGFPC1-huNFATc1EE-WT [63 (link)] plasmids and then plated on polylysine-coated coverslips. Forty-eight hours post-transfection cells were stimulated with 2 mM CaCl₂ modified Ringer’s solution with 2 μM thapsigargin (TG) (Merck, Darmstadt, Germany, Cat#67526-95-8) for 45 min. Cells were fixed with a fixative solution [4% w/v formaldehyde (freshly prepared from paraformaldehyde, Sigma-Aldrich, San Louis, MO, USA, Cat#158127), 4% w/v sucrose (Sigma-Aldrich, Cat#S0389) in Dulbecco’s phosphate-buffered saline (DPBS), at pH 7.4. After three washes for 10 min each, Hoechst 33342 nuclear stain at 200 ng/mL (Thermo Fisher Scientific, Waltham, MA, USA, Cat#H3570) was performed. Samples were mounted with Fluoromount (Sigma-Aldrich, San Louis, MO, USA, Cat#F4680). Images were acquired with 63× objective 1.35 N.A. using optical sectioning and structured illumination Zeiss Apotome 2, Axiovert 7.