Sp5 software las af

Paraffin-embedded sections of spleens were de-paraffinized with Xylene and hydrated in declining concentrations of ethanol. Slides were then rinsed in PBS and covered with Proteinase K (20 mg/ml in 50 mM Tris, 1 mM EDTA, 0.5% Triton X100, pH = 8.0) for 3 min at RT. Sections were blocked with 2% Goat serum for 30 min at RT and stained overnight at 4 °C, with Rat anti Mouse CD138 antibody (Biolegend, San Diego, CA, USA) diluted 1:400, followed by Goat anti-Rat IgG (H+L), Alexa Fluor^® 555 (Thermo Fisher Waltham, MA, USA) diluted 1:500 and DAPI (1 μg/μl) for 1 hour at RT. Coverslips were mounted and the images were viewed with a  ×  63/1.4 oil objective, using a Leica TCS SP5 microscope and Leica SP5 software (LAS-AF, Leica, Germany).

BMDM (5 × 10⁵ cells) were incubated in 24 wells and stimulated in the presence or absence of EPO 5 U/ml for 24 h. 5T33MM (1.5 × 10⁵ cells) stained with carboxyfluorescein succinimidyl ester (CFSE) were added to each well. The plates were spun-down and incubated for 24 hours at 37 °C. Cells were collected, stained with anti-mouse CD11b PE (eBioscience) and analyzed by FACSort flow cytometery (Becton-Dickinson); phagocytosing BMDM were positive for both CFSE and PE. Phagocytosis was also assessed by microscopy using a Leica TCS SP5 microscope and Leica SP5 software (LAS-AF, Leica, Germany).

HSC-T6 cells were incubated in a 15 mm² cell culture flask with nonantibiotic DMEM overnight, Hank’s wash was used twice, and then the cells were incubated with Fluo-3/AM (4 µM) and Pluronic F-127 (0.02%) at 37°C for 30 min. After washing three-times with Hank’s solution to remove the extracellular Fluo-3/AM, cells were placed onto the stage of confocal laser scanning fluorescence microscopy and analyzed by Leica-sp5 LAS AF software.