Anti cd34 fitc

ADSCs expanded to passage four were examined for surface marker expression using flow cytometry. The following monoclonal antibodies conjugated to fluorochromes were used: anti-CD13-APC, anti-CD29-PE, anti-CD31-FITC, anti-CD34-FITC, anti-CD44-APC, anti-CD45-FITC, anti-CD49a-PE, anti-CD63-FITC,-anti-CD73-PE, anti-CD90-APC, anti-CD105-FITC, anti-CD106-APC and anti-CD-166-PE (all from Becton Dickinson, Heidelberg, Germany). Isotype antibodies were included for all fluorochromes.
Cells were detached with 0.25% trypsin-EDTA, incubated with directly conjugated MAbs in FACS-buffer (1% FCS, 0.1% NaN₃ in PBS) for 30 minutes on ice, washed twice with FACS buffer, and fixed with 1% paraformaldehyde/PBS. Cells were analyzed using a FACSCanto flow cytometry system (Becton Dickinson). Data acquisition was performed with Diva software (Becton Dickinson) and data were analyzed using FCS express V3 (De Novo Software).

Cryopreserved PBMCs and their subpopulations were analysed with a 28-color flow cytometry panel, as previously described [34 (link)]. The following mAbs were used: anti-CD20 BUV805, anti-CD10 APC, anti-Vαβ TCR BUV737, anti-CD4 APCCy7, anti-CD25 PECy7, anti-CD27 PECy7, anti-CD27 PE, anti-CD45RA PerCpCy5, anti- CXCR5 BUV615, anti-IgG APC, anti IgG BB660, anti-IgD BV480, anti-IgG BV605, anti-IgA1/A2 PECy5, anti-CD8 BUV496, anti-CD21 BUV563, anti-PD1 BV605, anti-IgM PerCPCy5.5, anti-IgM APC R700, anti-CD3 BV421, anti-IL-2 BV711, anti-IL-9 PerCPCy5.5, anti-IL-13 BV421, anti-IL-17F BV786, anti-IFN-γ BV605, anti-TNF-α BUV395, anti-CD19 BV711, anti-CD34 FITC, anti-CCR6 PE, anti-CD45RA BUV395, anti-CXCR5 BV615 (all from Becton Dickinson); anti-CD20 Pacific Blue, anti-CCR7 PECy7, anti-CD127 BV650, anti-IL-17A APCCy7, anti-CD20 BUV805, anti-CXCR3 BV421, anti-CD3 BV570 (BioLegend); (OptiBuild); anti-CCR7 FITC (R&D Systems); anti-IL-4 PECy7, anti-IL-21 e660, anti-IL-22 PE (Thermo Fisher Scientific).

Cells (CML cell lines and primary cells) were incubated with increasing concentrations of nilotinib for 2 h. In primary cell samples, lymphocytes, monocytes, and polymorphonuclear cells were identified on the basis of their forward and low side light scattering characteristics. At least 50 000 target events were acquired and analysed. To analyse CD34⁺ cells, samples were first incubated with 5 µl of anti-CD34-FITC and 5 µl of anti-CD38-PE antibodies (Becton Dickinson, Le Pont de Claix, France) at room temperature for 20 min, and then washed once in 1 mL of PBS/1% bovine serum albumin.

The 3rd–5th passages of MSCs were harvested by trypsinization and incubated with 10 μL of the following mouse anti-human antibodies: anti-CD34-FITC (BD Pharmingen, USA), anti-CD45-PE (BD Pharmingen, USA), anti-CD90-FITC (AbD Serotec, USA), anti-CD73-PE (BD Pharmingen, USA), and anti-CD105-PE (Miltenyi Biotec, Germany) for 30 minutes at 4°C in the dark. After incubation, cells were washed twice with PBS and fixed with 300 μL 1% (v/v) paraformaldehyde. The expression profiles of cell surface markers were then determined by FACSCalibur flow cytometry (Becton Dickinson, USA) using CellQuest software. Cells labeled with FITC-conjugated mouse IgG1 (eBioscience, USA) and PE-conjugated mouse IgG1 (eBioscience, USA) served as negative controls.

Single-cell suspensions from bone marrow or spleen were prepared by passing the tissues through 70 μm cell strainers (BD Pharmingen, San Diego, CA) and red blood cells were lysed in RBC lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA). One million cells from each sample were then labeled with the following, anti-CD41-FITC (BD Pharmingen) or anti-TER119-APC-Cy7 (BD Pharmingen) for 30 minutes on ice. Labeled cells were washed in autoMACS Rinsing Solution (Miltenyi Biotec, Aubum, CA) with the addition of 0.5% BSA, fixed with 1% paraformaldehyde in PBS overnight and then analyzed on a LSRII cytometer (BD Biosciences, San Jose, CA). For analysis of Lineage^-Sca1⁺c-Kit⁺ (LSK) cell and myeloid progenitor cells, ten million bone marrow cells were lineage depleted by using the Lineage Cell Depletion Kit (Miltenyi Biotec) according to the manufacturer's protocol with the addition of the anti-IL-7R-biotin antibody (BD Pharmingen). After magnetic separation of the lineage cells through the MACS MS column, the lineage negative cells were incubated with streptavidin-PE-Cy5.5 (eBioscience, San Diego, CA), anti-Sca1-PE-Cy7 (BD Pharmingen), anti-c-Kit-APC (BD Pharmingen), anti-FcγR-PE (BD Pharmingen) and anti-CD34-FITC (BD Pharmingen) on ice for 30 minutes and analyzed on a LSRII cytometer after washing and fixation as described above.

DZNeP (Cat.No S7120) and Ven (Cat.No S8048) were purchased from Selleck (Shanghai, China). For in vitro experiments, DZNeP and Ven were dissolved in anhydrous dimethyl sulfoxide (DMSO). IMDM was taken from HyClone Cytiva (Shanghai, China), and other mediums and FBS were obtained from Gibco (Beijing, China). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). For flow cytometry antibodies, mouse anti-human MAbs and apoptosis antibodies were purchased from BD Biosciences (BD, USA), including anti-CD34-FITC, anti-CD117-PE, the FITC Annexin V apoptosis Detection Kit, APC Annexin V, and 7-AAD. The antibodies for the western blot were cleaved caspase-3 (9664, CST), PARP (9542, CST), BCL-2 (Ab32124, Abcam), BAX (Ab32503, Abcam), MCL1 (66026-1-Ig, Proteintech), PIK3IP1 (NBP1-69623, Novusbio), and GAPDH (Ab181602, Abcam). The PCR primers of EZH2, phosphoinositide-3-kinase-interacting protein1 (PIK3IP1), and GAPDH were synthesized by Sangon Biotech (Shanghai, China). The shRNA plasmid to PIK3IP1 was purchased from Corues Biotechnology (Nanjing, China). The ExFect transfection reagent was obtained from Vazyme (Nanjing, China).