BD Pharmingen™ DAPI Solution

Panel 1. Two-color flow cytometric analysis of Jurkat cell viability. Jurkat cells were treated with DMSO vehicle (Left Plot) or 5 μM Camptothecin (Right Plot) overnight. Cells were resuspended in Annexin V Binding Buffer (Cat. No. 556454) and stained with FITC Annexin V (Cat. No. 556419) and 0.06 μg/mL DAPI. Camptothecin-treated cells show an increased frequency of apoptotic (Annexin V+DAPI-) and dead (Annexin V+DAPI+) cells. Analysis was performed using a BD LSRFortessa™ Cell Analyzer System. Panel 2. Flow cytometric analysis of HeLa cell DNA content. Cultured HeLa cells in log phase growth were harvested using Gibco® Cell Dissociation Buffer (Life Technologies), fixed, and permeabilized using BD Pharmingen™ Transcription Factor Buffer Set (Cat. No. 562574/562725). Cells were resuspended in DPBS with 1 μg/mL DAPI and analyzed using a low BD LSRFortessa™ cytometer flow rate. A 405 nm laser with a 450/50 nm bandpass filter was used to collect data; comparable results were obtained using a 355 nm laser with a 450/50 nm bandpass filter (not shown). Histograms were deconvoluted by FlowJo™ software into G0/G1, S, and G2/M populations. Panel 3. Multicolor immunofluorescence analysis of NKX2.2 and PTF1A expression in mouse pancreas. Following antigen retrieval with BD Retrievagen A Buffer (Cat. No. 550524), a formalin-fixed paraffin-embedded pancreas tissue section was stained with Purified Mouse Anti-Nkx2.2 antibody (Cat. No. 564731, pseudo-colored red), washed, and stained with Alexa Fluor® 488 Goat Anti-Mouse Ig (Life Technologies). After washing, the section was stained with Alexa Fluor® 647 Mouse Anti-PTF1A (pseudo-colored green) and counterstained with DAPI (pseudo-colored blue). The nuclei of islet cells (DAPI+) stain positively for both NKX2.2 and PTF1A. The nuclei of acinar cells and some islet precursor cells stain positive for PTF1A. All images were analyzed using a BD Pathway™ 435 High-Content Bioimager System and merged with BD Attovision™ Software.