PDLSCs were grown in GM and detached using 1 mM EDTA. Upon harvesting, cells were washed in cold Dulbecco’s phosphate-buffered saline (PBS; Capricorn) supplemented with 0.5% bovine serum albumin (BSA; Sigma-Aldrich, Saint Louis, MO, USA) and separated into aliquots of 2 × 105 cells. Cells were then labeled for 30 min at +4 °C in the dark with monoclonal antibodies specific for human antigens including CD45, CD235a, CD90, CD44H, CD73 (all from R&D Systems, Minneapolis, MN, USA), CD-105, CD29 (Invitrogen, Waltham, MA, USA), CD34 (Dako Cytomation, Glostrup, Denmark) and CD11b (Biosource, Camarillo, CA, USA), each of which was conjugated with either FITC or PE. To determine the level of nonspecific binding, FITC- and PE-conjugated isotype control antibodies (R&D Systems) were used. The analysis was conducted using a CyFlow SL flow cytometer (Partec, Münster, Germany).
Fitc and pe conjugated isotype control antibodies
Manufactured by R&D Systems
FITC- and PE-conjugated isotype control antibodies are laboratory reagents used to establish background fluorescence levels in flow cytometry experiments. These antibodies are designed to bind non-specifically to cells, providing a negative control to differentiate specific from non-specific antibody binding.
Automatically generated - may contain errors
2 protocols using fitc and pe conjugated isotype control antibodies
1
Phenotypic Characterization of PDLSCs
2
Phenotypic Characterization of ASCs
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ASCs were maintained in standard conditions and after reaching confluence were harvested using 1 mM EDTA and washed in cold PBS supplemented with 0.5% BSA. Cells were incubated (30 min in the dark at 4 C) with fluorescein isothiocyanate (FITC)-or phycoerythrin (PE)-conjugated antibodies against human antigens CD44H, CD73, CD90, (all from R&D Systems, Minneapolis, MN), CD105 (from Invitrogen, Carlsbad, CA) and CD34 (Dako Cytomation, Glostrup, Denmark). The percentage of non-specific binding was determined by using the appropriate FITC-and PE-conjugated isotype control antibodies (R&D Systems, Minneapolis, MN). Flow cytometry was performed using flow cytometer Partec (Munster, Germany).
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