Apc conjugated anti cd45

EPCs were digested with 0.25% trypsin, washed three times with PBS, and incubated for 30 min on ice with 100 μl PBS containing the surface marker antibodies such as FITC-conjugated anti-CD133, APC-conjugated anti-CD34, PE-conjugated anti–cluster of differentiation 31 (CD31), FITC-conjugated anti-CD14, APC-conjugated anti-CD45, and anti-kinase insert domain receptor (KDR) (BD Biosciences, San Jose, CA, USA). Quantitative flow cytometry was performed using FACS Calibur (BD Biosciences), and data were analyzed using FlowJo software (BD Biosciences).

Cell concentration was counted using a cell chamber. For the quantification of cell types among the isolated cells, the cells were immunophenotyped using a panel of fluorescence-conjugated antibodies. The cells were incubated with Fc- receptor blocking solution (Miltenyi) to block unspecific binding of antibodies, followed by incubation with specific antibodies according to manufacturer recommendations. Panel of used fluorescence-conjugated antibodies in the study as followed: PE-conjugated-anti-CD44 (Beckman Coulter, cat.no: A32537), PE-conjugated-anti-CD90 (Beckman Coulter, cat.no: IM3600U), PE-conjugated-anti-CD105 (Beckman Coulter, cat.no: A07414), FITC-conjugated-anti-CD271 (Biolegend, cat.no: 345104), FITC-conjugated-anti-CD31 (BD Pharmingen, cat.n. 555445), PE-conjugated-anti-CD34 (BD Pharmingen, cat.no: 555822), PE-conjugated-anti CD14 (BD Pharmingen, cat.no: 555398) and APC-conjugated-anti-CD45 (BD Pharmingen, cat.n. 555485), FITC-LEPR (R&D systems, cat.n. FAB867F), PE-SOX2 (R&D systems, cat.n. IC2018P), PE-CD49a (BD Pharmingen, cat.n. 559596). Following incubation for 30 min in the dark at 4 °C, cells were washed and analysed by BD LSR II (BD Biosciences). The flow cytometry analysis was performed by Kaluza 1.1 analysis software. Flow cytometric gating was defined based on relevant isotype controls.

The MSCs were stained using the following mouse monoclonal antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-human CD90, phycoerythrin (PE)-conjugated anti-human CD105, allophycocyanin (APC)-conjugated anti-human CD73, FITC-conjugated anti-human CD44 (BD Biosciences), PE-conjugated anti-HLA-ABC (BD Biosciences), FITC-conjugated anti-HLA-DR (BD Biosciences), FITC-conjugated anti-human CD34 (BD Biosciences), PE-conjugated anti-human CD 11b (BD Biosciences), PE-conjugated anti-human CD 19 (BD Biosciences), and APC-conjugated anti-CD45 (BD Biosciences). Propidium iodide was used to identify and exclude the dead cells. The stained cells were acquired with a FACS Canto II flow cytometer (BD Biosystems) and analyzed using the FlowJo software (Tomy Digital Biology, Co. Ltd.). As for microglia, EYFP-expressing microglia were stained using APC-conjugated anti-mouse CD86 (BioLegend), PE-conjugated anti-mouse CD206 (ThermoFisher), and the Live/Dead™ fixable near-IR dead cell stain kit (Invitrogen). The stained cells were acquired with a Gallios flow cytometer (Beckman Coulter) and analyzed using the FlowJo software (Tomy Digital Biology, Co. Ltd.). Three independent FACS experiments were performed.

Aliquots of 10⁶ cells from the peritoneal cavity were transferred to 1X
Hank’s Salt Solution supplemented with 15 mM HEPES pH 7.4, 5% fetal bovine
serum, 0.5 U/ml DNase I. After blocking with FcBlock (antiCD16/CD32, BD Biosciences,
San Jose, CA), cells were incubated with APC conjugated anti-CD45 for 20 min and
washed (BD Biosciences, San Jose, CA). Cell samples were analyzed in a FACSCalibur,
where we used the FL2 channel to determine cells autofluorescence and FL4 to detect
cells stained with anti-CD45. We determined the absolute numbers of L929 cells in the
peritoneal cavity of each mouse by multiplying the percentage of CD45- cells in each
sample by the total number of cells in the peritoneal lavage.

Healthy female hamsters, 8 weeks old, were euthanized and femurs and tibias were procured under sterile conditions. The epiphyses were removed and bone marrow cells were collected by flushing bones with sterile phosphate buffer saline (Gibco, Auckland, New Zealand). Recovered cells were resuspended in alpha-MEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 80 μg/ml gentamycin (Sanderson Laboratory, Chile), and plated at a density of 0.25 × 10⁶ nucleated cells/cm² in plastic tissue-culture dishes. After 72 h, nonadherent cells were removed by medium change. When foci reached confluence, adherent cells were detached with 0.25% trypsin, 2.65 mM EDTA (Gibco) and subcultured. At 70–80% confluence, cells were tripsinized, centrifuged, resuspended in alpha-MEM, counted, characterized, and injected.
Cell immunophenotyping was performed by flow cytometry after immunostaining with APC-conjugated anti-CD45 (BD Pharmingen, USA), FITC-conjugated anti-alpha smooth muscle actin (Sigma), and FITC-conjugated mouse anti-vimentin (Oncogen, USA) [2 (link)]. To assess differentiation potentials, cells were incubated with adipogenic or osteogenic induction media [20 (link)]. Seven and 14 days later, samples were stained with Oil Red O or Alizarin Red (Sigma) (Additional file 1: Figure S1).