Anti cd34 fitc and pe

At confluence, the cells derived from the Rigenera digestion method or the lipoaspirates from the digestive solution were detached with trypsin-EDTA (200 mg/L EDTA, 500 mg/L trypsin; Cambrex, Milan, Italy). At least 200,000 cells were, directly placed in incubation using fluorescent conjugated antibodies for 30 min at 4 °C then washed and re-suspended in 0.6 mL. Specimens were examined using FACS Aria II flow cytometry (Becton & Dickinson, Mountain View, CA, USA). The antibodies herein investigated were: anti-CD117 PE (c-kit) (Miltenyi-Biotech, Calderara di Reno, Bologna, Italy); anti-CD34 FITC and PE (Miltenyi-Biotech); anti-CD90 FITC (BD Pharmingen, Buccinasco, Milano, Italy); anti-CD105 FITC (Santa Cruz, CA, USA); anti-CD73 PE (Miltenyi-Biotech); anti-CD29 PE (Miltenyi-Biotech); anti-CD31 FITC (Miltenyi-Biotech); and anti-CD45 Cy and PE (BD Pharmingen).

After digestion, at least 500,000 fresh cells were incubated with primary antibody for 30 min at 4 °C, washed twice in PBS and incubated with a secondary antibody. Antibodies used in this study were as follows: anti-CD117 PE (c-kit) (Miltenyi-Biotech, Calderara di Reno, Bologna, Italy—catalog number 130-098-212), anti-CD34 FITC and PE (Miltenyi-Biotech, Calderara di Reno, Bologna, Italy—catalog number 130-098-142 and 130-098-140), anti-CD90 FITC (BD Pharmigen, Buccinasco, Milan, Italy—catalog number 555595), anti-CD105 FITC (Santa Cruz, CA, USA—catalog number sc-18838), anti-CD29 Cy (Miltenyi-Biotech, Calderara di Reno, Bologna, Italy—catalog number 130-101-281), anti-CD31 FITC (Miltenyi-Biotech, Calderara di Reno, Bologna, Italy—catalog number 130-098-171), anti-CD133 PE (Miltenyi-Biotech, Calderara di Reno, Bologna, Italy—catalog number 130-098-826), anti-CD73 PE (BD Pharmigen, Buccinasco, Milan, Italy—catalog number 550257), anti-CD44 FITC (Miltenyi-Biotech, Calderara di Reno, Bologna, Italy—catalog number 130-098-210), anti-CD45 Cy and PE (BD Pharmigen, Buccinasco, Milan, Italy—catalog number 560974 and 555483), and anti-CD14 PE (Miltenyi-Biotech, Calderara di Reno, Bologna, Italy—catalog number 130-098-067). A FACS Vantage (Becton Dickinson) was used. CellQuest software was used for analyses.