Rabbit igg anti c kit

After seeding hDPSCs at a density of 1.5 × 10³ cell/cm² on titanium disks in 48-multiwell units and after 48 h of culture on each disk, cells were fixed in 4% ice-cold paraformaldehyde in PBS for 15 min and then processed as described above. The presence of the stemness markers STRO-1 and c-Kit was evaluated by immunofluorescence analysis in which the following primary Abs diluted 1:100 were used: mouse IgM anti-STRO-1 and rabbit IgG anti-c-Kit (Santa Cruz, Dallas, TX, USA). Secondary Abs (goat anti-mouse IgM Alexa488, goat anti-rabbit Alexa546) were diluted 1:200 (Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were stained with 1 µg/mL 40,6-diamidino-2-phenylindole (DAPI) in PBS 1%. The multi-labeling immunofluorescence experiments were carried out avoiding cross-reactions between primary and secondary Abs. Confocal imaging was performed using a Nikon A1 confocal laser scanning microscope, as previously described. The confocal serial sections were processed with ImageJ software (NIH, Bethesda, MD, USA) in order to obtain 3-dimensional projections and image rendering was performed by Adobe Photoshop Software.

In order to assay the percentage of cells expressing STRO-1, c-Kit and CD34 surface antigens, FACS analysis was performed on the whole unsorted hDPSCs after in vitro expansion (~5 × 10⁶ cells), at passage 1. Likewise, the percentage of cells triple labelled for STRO-1, c-Kit and CD34 or double labelled for STRO-1, c-Kit and negative for CD34 expression was evaluated.
Indirect staining was performed using mouse IgM anti-STRO-1, rabbit IgG anti-c-Kit (Santa Cruz) and mouse IgG anti-CD34 (Millipore), followed by goat anti-mouse-IgM-Alexa488, donkey anti-rabbit-IgG-Alexa647, and goat anti-mouse-IgG-Alexa405 (Invitrogen). Non-specific fluorescence was assessed by using normal mouse IgG or IgM followed by the secondary antibody as described above. Cells were acquired using Attune acoustic flow cytometer (Life Technologies, Thermo Fischer Scientific) equipped with four lasers (blue 488 nm, yellow 561 nm, red 638 nm and violet 405 nm). Data were analyzed using FlowJo 9.8 (Treestar, Miltenyi).

After 24 h and 4 days of culture on each disk, respectively, cells were fixed in 4% paraformaldehyde in PBS for 15 min and then processed as previously described [39 (link)]. The following primary antibodies were diluted 1:50 in bovine serum albumin 1% (BSA): mouse IgM anti-STRO-1 and rabbit IgG anti-c-Kit (Santa Cruz, Dallas, TX, USA). Secondary antibodies (goat anti-mouse IgM AlexaFluor488, goat anti-rabbit AlexaFluor546) were diluted 1:200 in 1% BSA (Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were stained with 1 μg/mL DAPI in 1% PBS for 5 min, then disks were mounted with 1,4-diazabicyclo [2.2.2] octane solution (DABCO, Sigma Aldrich, Saint Louis, MO, USA) anti-fading medium. The multi-labelling immunofluorescence experiments were performed avoiding cross-reactions between primary and secondary antibodies. Confocal imaging was done with a Nikon A1 confocal laser scanning microscope. Fiji ImageJ software (NIH, Bethesda, MD, USA) was used in order to process confocal serial sections and to obtain 3-dimensional projections, while image rendering was performed by Adobe Photoshop Software (Photoshop 7.0, Adobe, San Jose, CA, USA) [52 (link)].