Anti stro 1

For flow cytometry, the adherent cells were removed by trypsinization,
washed with Ca²⁺, Mg²⁺-containing 1× phosphate
buffered saline (PBS) and blocked with 3% fetal bovine serum (FBS) in PBS for 30
min at 4°C. Cells were aliquoted (100 μl/tube) for binding and
staining with 2.5 μg/ml of FITC- or Alexa Fluor (AF)-conjugated
antibodies. Sorting was performed using a BD Biosciences fluorescence-activated
cell sorting (FACS) DiVa High-Speed Cell Sorter (San Diego, CA) with 350, 488,
and 633 nm lasers. Anti-CD34 to Alexa Fluor 488 and anti-Stro-1 were conjugated
to Alexa Fluor 488, anti-CD45 to FITC, anti-CD90 to PE, anti-CD105 to Alexa
Fluor 488, and to the appropriate isotype controls (all from BD Pharmingen Inc.,
San Diego, CA except anti-Stro-1, which was obtained from Invitrogen, Carlsbad,
CA). The data were analyzed using FlowJo software (Tree Star, San Carlos, CA).
Propidium iodide was used to exclude dead cells, and percentages of positively
stained-cells were calculated by subtracting the value of isotype controls.
Cells were negatively selected for CD34 (NOVUS, catalog# NB600–107 AF
488) and CD45 (BD Pharmingen, Catalog# 554877 FITC) and positively selected for
CD90 (BD Pharmingen Catalog# 554898 PE), CD105 (Application.Inc, catalog#
CA1725), and Stro-1 (Invitrogen, catalog# 398407).

Adherent BMSCs cells at P3 in T75 flasks were removed by trypsinization, washed with Ca²⁺, Mg²⁺-containing 1X PBS and blocked with 3% FBS in PBS for 30 min at 4°C. Cells were aliquoted (100 μl/tube) for binding and staining with 2.5 μg/mL of FITC- or Alexa Fluor (AF)-conjugated antibodies. Sorting was performed using a BD Biosciences FACS DiVa High-Speed Cell Sorter (San Diego, CA) with 350 nm, 488 nm, and 633 nm lasers. Anti-CD31, anti-CD73, and anti-Stro-1 were conjugated to Alexa Fluor 488, anti-CD45 to FITC, anti-CD90 to PE, anti-CD105 to Alexa Fluor 647, and to the appropriate isotype controls (all from BD Pharmingen Inc., San Diego, CA except anti-Stro-1, which was obtained from Invitrogen, Carlsbad, CA). Analysis was performed on a FACS Aria III BD (Becton Dickinson, Franklin Lakes, NJ), and the data were analyzed using FlowJo software (Tree Star, San Carlos, CA). Propidium iodide was used to exclude dead cells, and percentages of positively stained-cells were calculated by subtracting the value of isotype controls. Cells were negatively selected for CD31 and CD45 and positively selected for Stro-1, CD73 and CD105 binding. Cells were collected in 1 mL DMEM supplemented with 10% FBS and cultured and passaged to P3 for further studies.