The positive expression of surface and intracellular markers was confirmed by confocal microscopy in both rAT-MSCs and hAT-MSCs. have, despite the slight differences in marker expression, the comparable phenotype as human AT-MSCs and possess the neurodifferentiation ability. Accordingly, rAT-MSCs should be subjected to further studies with potential application in veterinary medicine but also, in case of their cryopreservation, as a source of genetic information of endangered species stored in the gene lender. for 5 min. 2.4. Isolation and Culture of Rabbit Stem Cells Adipose tissue samples were incubated at 37 C for about 2 h with collagenase type I (Sigma Aldrich, Gillingham, UK) at a concentration of 0.2%. The enzymatic answer was neutralized with a culture medium and filtered through a 100 m filter to remove the undigested tissue. After filtration, the samples were centrifuged at 1200 for 10 min. Following centrifugation, cell pellets were resuspended in GibcoTM MEM culture medium (Thermo Fisher Scientific) supplemented with 20% fetal bovine serum (Sigma Aldrich, Gillingham, UK) and 1% penicillin/streptomycin antibiotics (Thermo Fisher Scientific). The medium was changed every 3 days to remove non-adherent cells. Stem cells isolated from your adipose tissue (AT-MSCs) reached 90% confluency in about 6C7 days after isolation. Cells were cultured until passage 3 (P3), as previously described . Isolation and culture Saikosaponin B2 of stem cells from your amniotic fluid (AF-MSCs) and the bone marrow (BM-MSCs) were described in previous studies [31,32,33]. Briefly, amniotic fluid was diluted (1:1) with a culture medium; EBM-2 basal medium (Lonza, Walkersville, MD, USA) supplemented with 20% fetal bovine serum (Sigma Aldrich), EGM-2 SingleQuots? Kit (Lonza), and Saikosaponin B2 1% penicillin/streptomycin. Femoral bone heads were removed under sterile conditions and bone marrow was flushed using PBS (without Ca2+ and Mg2+ ions). After filtration the cell suspension was layered on a Biocoll (Biochrom, Berlin, Germany) and separated using density gradient centrifugation at 867 and 20 C for 20 min. Density of cell seeding was as follows: 1.2 104 cells/cm2 for amniotic fluid and adipose tissue and 1.2C1.5 106 cells/cm2 for the bone marrow. All types of rabbit stem cells were maintained under the same conditions at 37 C and a Saikosaponin B2 5% CO2 in the atmosphere. 2.5. Culture of Human Adipose-Derived Stem Cells Commercially available human AT-MSCs (hAT-MSCs; C-12977, PromoCell, Heidelberg, Germany) were obtained at passage 2. Cells were cultured in GibcoTM MEM culture medium (Thermo Fisher Scientific) supplemented with 20% of fetal bovine serum (Sigma Aldrich) and 1% of antibiotic/antimycotic answer (Biowest). Cells were seeded on 75 cm2 culture flasks at a density of 1 1.2 104 cells/cm2 and maintained under standard conditions at 37 C and a 5% CO2 in the atmosphere. 2.6. Populace Doubling Time In order to determine the populace doubling time (PDT), cells were counted at every passage (P1CP3) and culture time was recorded. Cells were dissociated and concentration was counted as we described in our previous study . Populace doubling time was counted for each passage by the growth curve using the doubling time calculator available at http://www.doubling-time.com/compute.php (5 December 2020). 2.7. Detection of Surface and Intracellular Markers Using Flow Cytometry To confirm the origin of rabbit BM-MSCs, AF-MSCs and AT-MSCs, the detection of the cell surface and intracellular markers was performed by an antibody immunofluorescent staining, as explained in our previous studies [31,32]. The cells were double-stained using a rat anti-mouse IgG1-PE fluorochrome-conjugated secondary antibody (clone X-56; Miltenyi Biotec, Bergisch Gladbach, Germany) or goat anti-mouse IgG-FITC polyclonal antibody (STAR117F, Bio-Rad, Hercules, CA, USA). A complete list of main antibodies with an indication of their reported reactivities, used in this study, is shown in Table 1. To exclude the lifeless cells from your analysis, samples were co-stained with lifeless cell marker such as 7-AAD (eBioscience, Wien, Austria). Cells were analyzed using a FACS Calibur ? device (BD Biosciences, San Jose, CA, USA) and Cell Mission Pro ? software (BD Biosciences). At least 50,000 events were analyzed for each sample. Unstained FMO (fluorescence minus one) samples were used as control Rabbit Polyclonal to ELL samples in order to gated the positive cells according to the increased fluorescent intensity. Table 1 List of main antibodies utilized for circulation cytometry. < 0.05; *** < 0.001; Concontrol (non-induced sample), Neuroneurodifferentiated sample; rAT-MSCsrabbit adipose tissue-derived mesenchymal stem cells; rAF-MSCsrabbit amniotic fluid mesenchymal.