Anti annexin 5 pe

Activation markers, namely CD25 and CD69 were analyzed as described previously (Stoszko et al., 2016 (link)). Briefly, CD4+ T cells were treated with DMSO, compound, or PMA/Ionomycin for 24 and 72 hours. Cells were collected, washed with PBS and stained for 30 min at 4 °C with α-CD25-APC (17–0259-42, eBioscience) and α-CD69-FITC (11–0699-42, eBioscience). Following two washes with PBS, cells were fixed with 1% HCHO at 4 °C and analyzed by flow cytometry with Becton Dickinson Fortessa instrument. To determine percent of apoptotic cells after treatment cells were stimulated for 24 and 72 hours and stained with anti-AnnexinV-PE (BD Biosciences, cat. 556454) in the presence of 2.5 mM CaCl₂ for 20 min at 4 °C. Cells were analyzed by Becton Dickinson Fortessa flow cytometer. Data represents the average of six experiments performed on cells from different healthy donors. Viability of ex-vivo infected primary CD4+ cells was determined by flow cytometry on the basis of forward versus side scatter analysis.

To monitor the differentiation status, cells were stained with the following antibodies: APC-CD11b (ThermoFisher, CD11b 05), APC-CD13 (ThermoFisher, MHCD1305), PE-CD14 (BD Pharmingen, 555398), PE-CD33 (BD Pharmingen, 555450). To measure apoptosis, cells were washed with PBS and incubated with anti– PE-Annexin V (BD Pharmingen, 556421) in the ANNEXIN-V binding buffer in a reaction volume of 100 μl for 15 minutes based on manufacturer’s instruction. DAPI was added prior to analysis. Cells were analyzed on a BD FACS LSR Fortessa instrument.
To monitor human leukemia cell engraftment, bone marrow cells from recipient mice were stained with APC-mouse CD45.1 (Biolegend 110720) and PerCP-Cyanine 5.5-human CD45 (eBioscience, 45-9459-42). Immunoblot analysis of engrafted human cells was performed using sorted human CD45 positive cells.