Igg1 fitc

Immediately after isolation, 2 µl mouse platelets were incubated with antibodies against IgG1-FITC (Milteny Biotec, Bergisch Gladbach, Germany, order no.: 130-098-847, 1:25) or CD62P-FITC (BD Biosciences, Heidelberg, Germany, Cat: 561923, 1:25). Human platelets were incubated with antibodies against FITC Mouse IgG1 (BD Biosciences, Cat: 555748, 1:25) or FITC Mouse Anti-Human CD62P (BD Biosciences, Cat: 555523, 1:25). Subsequently, 46 µl of FACs buffer (2 mM EDTA, 0.5 % FCS ad 100 ml PBS, pH 7.1) was added and the samples incubated for 30 min at 4 °C in dark. To study apoptosis, 2 µl of platelets were incubated with 5 µl Annexin V-FITC (Milteny Biotec, Order no.: 130-097-928) in 100 µl Annexin V Binding Buffer (Miltenyi Biotec, 20x Stock Solution) at RT for 30 min in dark. Immediately before analysis, 2 µl of 7-AAD (Miltenyi Biotec) was added. All samples were centrifuged at 300g for 10 min and the pellets were resuspended in 100 µl of FACs flow (BD FACSFlow). FACs analysis was instantly performed with a BD FACScan.

MFAT samples were treated with collagenase IV (Serva GmbH) to retrieve SVF and analyze its various cell components, in particular the ASCs, expressing CD105 + , CD73 + , CD90 + and CD44 + and negative for HLA-DR, CD34, CD31 and CD45; CD146 + pericytes, CD31 + endothelial cells, CD34 + hematopoietic cells. The standard labelling protocol was performed with the following fluorochrome-conjugated antibodies and isotypic controls: ASCs were marked with MSC phenotyping kit, human CD44 PE, CD45 PerCP, IgG1 PE, IgG1 FITC, IgG1 APC, and IgG2a PerCP (Miltenyi Biotec, Bergisch Gladbach, Germany), CD31PE, CD34FITC, CD146APC and CD90 PerCP (Biolegend, San Diego, CA, USA). About 10⁵ events/sample were used for capture with MACsQUANT10 and data were analyzed with MACs quantify software (Miltenyi Biotec, Bergisch Gladbach, Germany). The percentage of ASCs in the MFAT volume was determined, and then the number of ASCs for ml of MFAT was normalized on the absolute number of viable cells.

Cell surface markers of undifferentiated AF-MSCs were detected using the protocol from Glemžaitė and Navakauskienė [53 (link)] with fluorescein isothiocyanate (FITC)-conjugated mouse anti-human antibodies against CD44 (cat. no. 156-3C11; Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), CD34 (cat. no. 130-113-178; Miltenyi Biotec, Bergisch Gladbach, Germany), and CD90 (cat. no. 11-0909-42; Molecular Probes, Thermo Fisher Scientific, Hillsboro, OR, USA), PE-conjugated mouse anti-human against CD105 (cat. no. 12-1057-42; Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), and mouse IgG2A-FITC (cat. no. 130-113-833) Miltenyi Biotec, Bergisch Gladbach, Germany), IgG1-FITC (cat. no. RMG101), IgG2b-FITC (cat. no. IgG2b-FITC) (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), or IgG1-PE (cat. no. GM4993) (Molecular Probes, Thermo Fisher Scientific, Hillsboro, OR, USA) as isotype controls. The cell surface marker of differentiated AF-MSCs, CD172α (SIRPA, cat. no. 15-414), was detected using the same protocol and allophycocyanin (APC)-conjugated CD172α (SIRPA, cat. no. 17-1721-82) Monoclonal Antibody (eBioscience, Thermo Fisher Scientific, San Diego, CA, USA). Labeled samples were measured using the BD FACSCanto™ II flow cytometer and BD FACSDIVA™ software (BD Biosciences, San Jose, CA, USA).

The characterization of the mesenchymal cells was performed according to the following criteria.

  Morphological characterization: mesenchymal cells must be elongated, spindle-shaped, narrow, and fibroblast-like, with a central nucleus and abundant lysosomes [9 (link)].

  Characterization by flow cytometry: several membrane markers were used and detected by flow cytometry. Previously, cultured cells were trypsinized, centrifuged, and resuspended in 100 µL of a saline buffer. Subsequently, the cells were incubated with 10 µL of fluorochrome-coupled monoclonal antibodies for CD34, CD45, CD73, CD90, and CD105 and mouse isotype control antibodies (FITC-IgG1, PE-IgG1, APC-IgG1 PerCP, IgG1, and PerCP-IgG2a) from the phenotyping kit for mesenchymal stem cells (MSCs; Miltenyi Biotec, Bergisch Gladbach, Germany). Samples were processed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). A homogeneous population was characterized by presenting cells positive for CD73, CD90, and CD105 and negative for CD34 and CD45 [10 (link)].

Cells were separated using a FACSAria BD cell sorter with the following cell surface markers: EpCam, CD44 and CD24, which have been reported previously [5 (link)]. The antibodies where bought from Miltenyi Biotec (Bergisch Gladbach, Germany) and BD Biosciences (Carlsbad, CA, USA). Briefly, cells were detached with Accutase (Life Technologies, Carlsbad, CA, USA) and washed with PBS 1%. A total of 15 × 10⁶ cells were resuspended in a stained buffer (PBS with FBS 1%) and then were incubated with antibodies for 30 min at 4 °C. FITC-IgG1 and PE-IgG1 (Miltenyi Biotec; Bergisch Gladbach, Germany) were used as isotypes controls. CSCs (CD44+/CD24low) and non-stem cells (CD44+/CD24+) were sorted on a FACSAria (fluorescence-activated cell sorter, Becton Dickinson, Franklin Lakes, NJ, USA). For validation, cells were seeded in MammoCult (StemCell Technologies; Vancouver, Canada) for 24 h before analyses. RNA was isolated using Trizol (Life Technologies, Mexico City, Mexico) and RNA was reverse transcribed with a Maxima First strand cDNA synthesis kit (Thermo Fisher Scientific, Rockford, IL, USA). The expression level of stem cell markers (ALDH1A1, OCT4, ALDH1A3 and KLF4) and survivin were assayed by real-time PCR using the SYBR or TaqMan Master Kit (Applied Biosystems, Mexico City, Mexico) and PCRs were performed on the QuantStudio 7 (Applied Biosystems).