Infinicyt software version 2

After a washing in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and 0.09% azide (AZ; wash solution, WS), cells were resuspended in 100 μL WS. 1 × 10⁶ cells were incubated with an adequate concentration of antibodies (Table 4) for 15 min at RT in the dark. Samples were acquired on an FACSCanto II cytometer (Beckton Dickinson Biosciences, San Jose, CA, USA) equipped with FACSDiva software version 1.7 (BD Biosciences). Analyses were performed using Infinicyt software version 2.0 (Cytognos, Salamanca, Spain). Annexin V and 7-AAD staining (BD Bioscience, USA) were used to exclude nonviable cells. Unstained cells were used to assess background fluorescence. The results were expressed as the absolute cell count (dual-platform method). Experiments were performed in triplicate.

5 × 10⁵ HS-5 cells transfected with FAK-specific shRNA or control shRNA cells were seeded in the 6-well plates for 24 h until cell attachment was achieved. CD34+ HD-HSPCs were enriched from the BMMNCs after CD34-PE staining (BD Bioscience) by using immune-magnetic separation (EasySep PE selection kit) according to the manufacturer’s protocol. 1 × 10⁵ CFSE-stained CD34+ HD-HSPCs were added to each well. The co-cultures were performed in MyeloCult™ H5100 (StemCell Technologies, Grenoble, France) supplemented with 10⁻⁶ M hydrocortisone (StemCell Technologies) for 14 days. The half medium was changed at day 7. At days 5 and 14, both cells types were collected and counted with Trypan Blue Solution, 0.4%. Next, the HS-5 cells and CD34+ HD-HSPCs were evaluated by flow cytometry for viability (7-AAD staining), proportion of cell subpopulations, and HSPC differentiation. The antibodies used for HSPC staining are listed in Table 4. Data analysis was performed using Infinicyt software version 2.0 (Cytognos).

To perform the surface marker study, first, the absolute counts per µL of fresh leukocytes were determined by counting live (trypan blue-negative) cells in 200 μL of staining buffer in a Newbauer chamber. The mean and SD for live cell recoveries from individual cardiac tissues were (7.0 ± 2.0) × 10⁵/heart. Then, the harvested cells were incubated with different monoclonal antibodies in a dark room for 30 min at room temperature. All antibodies were directly labeled. Furthermore, a final volume of 100 μL per panel was injected on the BD FACSCanto flow cytometer (Becton Dickinson, San Jose, CA, USA) and analyzed using the Infinicyt software version 2.0 (Cytognos, Salamanca, Spain). Four different panels of antibodies were used to perform the phenotypic characterization of mononuclear cells according to the Euroflow multicolor panels (Table 1).