Fitch conjugated anti rabbit antibody

Hyaluronic acid (HA) with a weight-average molecular weight (MW) of Low (L) 200, Medium (M) 500, and High (H) 1435 kDa were kindly provided by Altergon Italia s.r.l.. Phosphate buffer saline (PBS) tablets without calcium and magnesium were obtained from MP Biomedicals Inc. hUCMSCs cells were extracted from the Wharton jelly of the umbilical cord kindly gifted by Ospedale Evangelico Betania (Naples, Italy), Human Fetal Lung Fibroblast Cells (MRC-5) were purchased from ATCC. Dulbecco’s Modified Eagle Medium (DMEM) (Microgem Italy). Small Airway Epithelial Cell Growth Medium (SAGM) (Lonza C-41 24) and Fetal Bovine Serum (FBS) were purchased from Lonza (Basel, Switzerland). Eagle’s Minimum Essential Medium (EMEM), Hyclone, (MA, USA). Penicillin, streptomycin (10,000 U/mL) from Invitrogen and Life Technologies (Carlsbad, CA, USA) were employed. Trypsin and Ethylenediaminetetraacetic acid (EDTA) were from HiMedia (Mumbai, India). Mitomycin C (MMC), Formalin, bovine serum albumin (BSA) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (USA). Pulmonary-associated surfactant protein C (SPC) rabbit antibody from Abcam. Fitch-conjugated anti-rabbit antibody (Millipore, Billerica, MA, USA). Human pulmonary surfactant associated protein A-B-C and D ELISA kits were obtained from Elabscience.

In order to assess the differentiation of hUCMSC cells, the qualitative expression of surfactant proteins C (SPC) was evaluated by immunofluorescence using SPC antibody. Then, 1 × 10⁴ hUCMSCs were seeded on a fluoro-dish-35 mm (World Precision Instruments, Inc.), HA solubilized in SAGM was used to feed cells while DMEM and SAGM media were used as control (CTR), cell media were changed every 3 days for 21 days of cell culture with fresh medium. After this time, hUCMSC cells were fixed in 10% Formalin for 1 h, permeabilized with 0.1% Triton X-100, blocked with 1% BSA and incubated with SPC rabbit antibody (Abcam) diluted in 1% BSA at 4 °C overnight. After washing with PBS three times, Fitch-conjugated anti-rabbit antibody (Millipore, Billerica, MA, USA) was added to the cells for 3 h at room temperature. Finally, Cell nuclei were stained with blue DAPI for 10 min at 37 °C. Samples were observed by a confocal microscope system (Leica TCS SP5 MP) with 63X oil immersion objectives. Images were acquired with a resolution of 1024 × 1024 pixel. To determine the quantitative expression of human pulmonary surfactant protein A-B-C and D the supernatants for analysis were collected after 21 days of exposure to the biomaterials, and then analyzed using SP A-B-C and D ELISA kits according to the manufacturer’s protocol.