Passage 3 eDPCs (2 × 105 cells) were resuspended in 500 µl of FACS buffer (PBS containing 1% sodium azide and 0.5% BSA, pH 7.2).
The cells were incubated with various antibodies, including CD11a/CD18 (Clone CZ3.2, 117, 2E11, B10; Gifts from Dr. Douglas Antczak, Cornell University, Ithaca,
NY, U.S.A.), CD34-FITC (Clone CA 581/CD34; BD Biosciences, San Jose, CA, U.S.A.), CD44 (Clone CVS18; AbD Serotec, Raleigh, NC, U.S.A.), CD45-FITC (Clone 2D1;
BD), CD90-FITC (Clone 5E10; BD), CD105-FITC (Clone SN6; Serotec), MHC Class I (Clone CZ3, 117, 1B12, C11; Gifts) and MHC Class II (Clone CZ11, 130, 8E8, D9;
Gifts) at 4°C for 30 min. All clones have been previously used in equine MSC experiments [15 (link),16 (link),17 (link),18 (link)]. The cells were washed twice with FACS buffer and
resuspended in 500 µl of FACS buffer. The cells incubated with the CD11a/CD18, CD44 and MHC class I and II antibodies were incubated with
anti-mouse IgG secondary antibodies labeled with FITC (Rockland, Gilbertsville, PA, U.S.A.) at 4°C for 30 min. Nonspecific FITC mouse immunoglobulin G1κ was
used as a negative control. Cells were then washed twice with FACS buffer and resuspended in 500 µl of FACS buffer. Cell fluorescence was
evaluated by flow cytometry with a FACSCalibur instrument (BD Biosciences). Data were analyzed using CellQuest Pro software (BD Biosciences).
The cells were incubated with various antibodies, including CD11a/CD18 (Clone CZ3.2, 117, 2E11, B10; Gifts from Dr. Douglas Antczak, Cornell University, Ithaca,
NY, U.S.A.), CD34-FITC (Clone CA 581/CD34; BD Biosciences, San Jose, CA, U.S.A.), CD44 (Clone CVS18; AbD Serotec, Raleigh, NC, U.S.A.), CD45-FITC (Clone 2D1;
BD), CD90-FITC (Clone 5E10; BD), CD105-FITC (Clone SN6; Serotec), MHC Class I (Clone CZ3, 117, 1B12, C11; Gifts) and MHC Class II (Clone CZ11, 130, 8E8, D9;
Gifts) at 4°C for 30 min. All clones have been previously used in equine MSC experiments [15 (link),16 (link),17 (link),18 (link)]. The cells were washed twice with FACS buffer and
resuspended in 500 µl of FACS buffer. The cells incubated with the CD11a/CD18, CD44 and MHC class I and II antibodies were incubated with
anti-mouse IgG secondary antibodies labeled with FITC (Rockland, Gilbertsville, PA, U.S.A.) at 4°C for 30 min. Nonspecific FITC mouse immunoglobulin G1κ was
used as a negative control. Cells were then washed twice with FACS buffer and resuspended in 500 µl of FACS buffer. Cell fluorescence was
evaluated by flow cytometry with a FACSCalibur instrument (BD Biosciences). Data were analyzed using CellQuest Pro software (BD Biosciences).
