Facs lyse wash assistant

Three isolates of cryopreserved BH and HAP ASCs were resuspended in low-glucose Dulbecco's modified Eagle's medium (Gibco® DMEM; Thermo Fisher Scientific, Frederick, MD) supplemented with either 10% FBS (GE Life Sciences; Logan, Utah) or 10% hPL. Cells were expanded to passage 2 or 3 (P2 or P3), trypsinized, washed, resuspended at 1 × 10⁶ cells/ml in HBSS, stained with mouse anti-human CD44, CD73, CD90, CD105, and CD142 (Human MSC Analysis Kit, BD Biosciences, San Jose, CA), and analyzed by fluorescence-activated cell sorting (FACS). 1 × 10⁵ cells were incubated with 1% BSA and 5 μl human Fc receptor blocking solution (BioLegend, San Diego, CA) for 5 min at room temperature (RT). Antibodies were added followed by 15 min of incubation at RT. Cells were then washed once in BD FACS Lyse Wash Assistant and analyzed on BD FACSCanto.

Differentiated cells were treated with 1X TrypLE™ Express for 5 min at 37 °C to obtain a single a cell suspension. Cell pellets were resuspended in FACS buffer (DPBS with FBS 2%) and cells were counted with a hemocytometer. A final amount of 1 × 10⁵ cells resuspended in 100 µl of FACS buffer with a dilution of 1:20 antibody was used for each analysis. The following cell surface antigens were analyzed for this study: KDR-PE (BD, 560494), CD34-APC (BD, 555824), CD43-FITC (Thermo Fisher Scientific, MHCD4301), CD41a-APCH7 (BD, 561422) and CD235a-BV421 (BD, 562938). Cells were washed using the BD FACS Lyse/Wash assistant (BD) and analyzed using BD LSRII flow cytometer (BD). Size and cell complexity were used to identify cell populations in a scatter graph representation. Single cells were discriminated using FSC-A and FSC-H and live cells were gated from single cell population using DAPI nuclear staining. Analysis of data was performed using FlowJo software (Tree Star Inc.). At least 10.000 events were collected for each analysis.

Cells were disassociated using Versene (EDTA) (Lonza), washed with PBS and fixed in paraformaldehyde (PFA; 2% final concentration in PBS) at 37 °C for 10 minutes. After washing with PBS, the cells were permeabilised with pre-chilled methanol (−18 °C) and incubated at 4 °C for 30 minutes, followed by a washing step. 0.2–0.5 × 10⁶ cells were resuspended in a total volume of 200 μl PBS containing 1% BSA and incubated with appropriate amounts of anti-CD44-BV421 (Cat.N.562890, BD Biosciences; 1:300 dilution), anti-TRA-1-60-FITC (Cat. N. FCMAB115F, Merck Millipore; 1:100 dilution) and Phospho-p38 MAPK (Cat. N. 4552, Cell Signaling Technology; 1:50 dilution; Mouse mAb Ig1 Isotype Control Alexa Fluor 647 Conjugate Cat. N.4843, Cell Signaling) monoclonal antibodies for 1 hour on a shaker, plate in the dark at room temperature. Finally, samples were washed using BD FACS™ Lyse Wash Assistant (BD Biosciences) and immediately analysed on a flow cytometer. FACS analysis was performed using BD FACS Canto II flow cytometer with FACSDiva software (BD Biosciences). A minimum of 20,000 events were recorded for each sample. Fluorescence minus One control (for each antibody) was used to gate the subpopulations.
Alkaline Phosphatase detection was performed with Alkaline Phosphotase detection kit (SCR004, Millipore) according to manufacture instructions.

Pluripotency marker expression was quantified with flow cytometric analysis. Single cell suspension was made with Versene® (EDTA) 0.02% solution and cells were fixed with 2% paraformaldehyde (PFA) for 10 minutes at 37 °C. Cells were then washed with PBS and re-suspended in ice cold methanol. They were then stored at −20 °C for at least 30 minutes and for up to 1 month. For the quantification of the expression of pluripotency markers, cells were washed with PBS, 1 × 10⁶ cells/ml were re-suspended in 100 μl of 2% FBS in PBS with the appropriate concentrations of antibodies, Anti-TRA-1-60, FITC conjugate (Merck Millipore; dilution 1:5) and Nanog XP® Rabbit mAb Alexa Fluor® 647 Conjugate (New England Biolabs; dilution 1:50). Cells were then incubated for 40 minutes on a shaker at room temperature. Cells were washed using BD FACS™ Lyse Wash Assistant (BD Biosciences) and analysed on BD FACSCanto™ II system (BD Biosciences). Data was analysed on BD FACSDiva software (BD Biosciences). A minimum of 10 000 events were recorded for each sample. Gating strategy involved using an appropriate isotype control.

Blood was collected from the retro-orbital sinus, facial vein, or tail vein of anesthetized mice to assess engraftment of human cells. Blood was lysed with the BD FACS Lyse Wash Assistant (BD Biosciences, San Jose, CA). Cells were stained with antibodies to human CD45 (BD Biosciences, # 561864, APC), mouse CD45 (BD Biosciences, # 559864, PE). Samples were assayed on the BD LSR II or LSR Fortessa (BD Biosciences) and data were analyzed with FlowJo version 9 (FlowJo, LLC, Ashland, OR). Samples were sorted for hCD45 and YFP on the BD Aria (BD Biosciences).

Citrated blood (500 µl) was immediately loaded into cryotubes containing 500 µl of 1% paraformaldehyde fixative solution. Paraformaldehyde-fixed blood (10 µl) was incubated with FITC-PAC-1 (15 min at room temperature). Following incubation, samples were further processed using lyse wash assistant (BD FACS Lyse Wash Assistant, BD Bioscience, CA, USA). Data acquisition was performed on BD FACS CANTO using FACSDiva software (BD FACSDIVA 6.0 software, BD FACSCanto Flow Cytometry, BD Biosciences, CA, USA). Process was repeated for isotype control antibody.