Fitc conjugated anti rabbit antibody

In order to assess the differentiation of hUCMSC cells, the qualitative expression of Surfactant Proteins C (SP−C) has been evaluated by immunofluorescence against SPC antibody. hUCMSCs were cultured for 21 days and seeded on fluorodish—35 mm (World Precision Instruments, Inc. Hitchin, UK), 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, Cambrige, UK) diluted in 1% BSA at 4 °C overnight. After washing with PBS three times, FITC-conjugated anti-rabbit antibody (Millipore, Billerica, MA, USA) was added to the cells for 3 h at room temperature. In parallel, qualitative expression of Cluster of Differentiation 73 (CD-73) has been evaluated by immunofluorescence against CD-73 antibody. hUCMSCs were cultured for 21 days and seeded on fluorodish—35 mm (World Precision Instruments, Inc.), were fixed in 10% Formalin for 1 h, permeabilized with 0.2% Tween 20 for 1 h, blocked with 1% BSA and incubated with CD-73 mouse antibody diluted in 1% PBS/BSA at 1:100 dilution for 2 h at 4 °C. Finally, for all samples, cell nuclei were stained with blue DAPI for 10 min at 37 °C. Samples were observed by confocal microscope system (Leica TCS SP5 MP) with a 63× oil immersion objective. Images acquired with a resolution of 1024 × 1024 pixels.

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), MRC-5 cells were purchased from ATCC. Dulbecco’s Modified Eagle′s—Medium (DMEM) (Microgem, Naples, Italy). SAGM (Lonza C-41 24) and Fetal Bovine Serum (FBS) were purchased from Lonza (Basel, Switzerland). 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). Formalin, bovine serum albumin (BSA) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). SPC rabbit antibody from Abcam. FITC-conjugated anti-rabbit antibody (Millipore, Billerica, MA, USA). CD-73 antibody (Sigma Aldrich, St. Louis, MO, USA). Human pulmonary surfactant associated protein (SP) SP-A, SP-B, SPC and D ELISA kits were obtained from Elabscience.

OC pieces were digested by 1 mg/mL collagenase and 1 μg/mL DNAse I (Sigma-Aldrich) for 120 min at 37 °C. Cell pellets were then suspended in running buffer (Miltenyi Biotec) and incubated with 10 µL of rabbit anti-human Ddx4 antibody for 30 min at 4 °C. Thus, the samples were treated with anti-rabbit IgG microbeads (Miltenyi Biotec), and Ddx4⁺cells were isolated by an automated magnetic-activated cell sorting system (autoMACS Pro, Miltenyi Biotec,) as previously described [11 (link)]. The purity of cell population was assessed by the flow cytometry detection of membrane Ddx4. Briefly, an aliquot of isolated cells was incubated with rabbit anti-human Ddx4 antibody (Abcam ab13840) and then processed with an FITC-conjugated anti-rabbit antibody (Sigma-Aldrich). In addition, cytoplasmic expression of Ddx4 was also evaluated by flow cytometry, after cell fixation and permeabilization as described for A2780 cells. In each case, an isotype control was used as negative parameter.