4 6 diamindino 2 phenylindole dapi

Bovine lactoferrin (bLf), derived from bovine colostrum, was provided as a generous donation by Fonterra, located in Palmerston North, New Zealand. Stearic acid (Grade I, with a purity of at least 99% according to gas chromatography) and a combination of high- and low-methoxyl pectin sourced from apples were used, as well as 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT), fluorescein isothiocyanate (FITC), fluorescein sodium salt, sodium azide, verapamil, MK-571 sodium salt hydrate, and ethylenediaminetetraacetic acid (EDTA), acquired from Sigma Aldrich, based in St. Louis, MO, USA. Soybean lecithin was obtained from BDH, located in Poole, UK. Poloxamer 188 was procured from BASF in Ludwigshafen, Germany. Chitosan with low viscosity was purchased from Fluka, situated in Park Rabin Rehovot, Israel. Dulbecco’s Modified Eagles’ Medium (DMEM), fetal calf serum, penicillin-streptomycin-glutamine, nonessential amino acids, trypsin-EDTA, sterile phosphate-buffered saline (PBS) (pH 7.4), and Hank’s balanced salt solution (HBSS) buffer (pH 7.4) were purchased from Life Technologies (Carlsbad, CA, USA). The 4′6-diamindino-2-phenylindole (DAPI) was purchased from Invitrogen (Auckland, New Zealand). The Caco-2 cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). All other reagents and chemicals were of analytical grade.

Cells were either allowed to settle briefly on poly-L-lysine coated chamber slides (VWR) at 37 °C or cytospun, fixed with 4% paraformaldehyde (USB Corporation), permeabilized with 1X Dako wash buffer (Dako) and blocked with 10–20% normal goat serum (Invitrogen) or donkey serum (Abcam) in 1X Dako wash buffer. Primary antibody incubation was overnight at 4 °C. The following primary antibodies were used: rabbit anti-Msi2 1:200 (Abcam), chicken anti-GFP 1:200 (Abcam) and mouse anti-Sdc1 1:100 (Abcam). Alexa fluor-conjugated secondary antibody incubation was performed for 1 h at room temperature. DAPI (4-6-diamindino-2-phenylindole; Molecular Probes) was used to detect DNA. Images were obtained with a Confocal Leica TCS SP5 II (Leica Microsystems).

The day prior to plating cells, 4-well removable chamber slides (Lab Tek II) were coated with Cell-Tak and kept at 4°C overnight. The next day, FLAG-tag MSCV-MSI2-IRES-GFP or FLAG-tag MSCV-MSI2HOXA9-IRES-GFP infected K562 cells were labeled with 100nM Mitotracker Deep Red FM (Thermo Fisher) at 37°C. After washing twice with PBS, cells were fixed with 4% PFA at 37°C and washed with warm PBS. Cells were spun onto the Cell-Tak coated chamber slides with the brake off and then allowed to settle for an additional 30 minutes. Cells were then permeabilized with 0.1% Triton X-100 for 3 minutes and then blocked in PBS with 10% normal goat serum, 5% bovine serum albumin, 0.3M glycine, and 0.05% Tween-20 for 1 hour. After blocking, cells were incubated with primary antibody in 1:10 diluted blocking buffer overnight at 4°C. The following primary antibodies were used: chicken anti-GFP 1.5:1000 (Abcam) and rabbit anti-FLAG 1.5:1000 (Cell Signaling Technology). Alexa fluor-conjugated secondary antibody (1:500) incubation was performed for 1 hour at room temperature. DAPI (4–6-diamindino-2-phenylindole; Molecular Probes) was used to detect DNA. Images were obtained with a Zeiss LSM-700 confocal microscope.