Journal: Apoptosis : an international journal on programmed cell death

Article Title: Recombinant human lactoferrin induces apoptosis, disruption of F-actin structure and cell cycle arrest with selective cytotoxicity on human triple negative breast cancer cells

doi: 10.1007/s10495-019-01539-7

Figure Lengend Snippet: The rhLf induces cytotoxicity on MDA-MB-231 cells in a dose-depended manner. Cells were exposed to a concentration gradient of rhLf and incubated for 24 h. Differential nuclear staining assay and a bioimager system were used to determine the cytotoxicity percentages (a; y-axis). When experimental samples were compared with solvent, 5% v/v H2O, (*) and untreated control cells (‡), the P values were of < 0.0001 and < 0.0001, respectively (a). Cells treated with 2 mM of H2O2 were used as positive control for cytotoxicity (a, e). Each experimental point represents the average of four replicas and error bars their corresponding standard deviation (a). A portion of representative live-cell images, merging the Hoechst and propidium iodide (PI) channels, used to obtain the cytotoxic activity are depicted in b–e. Nuclei exhibiting a blue signal are considered to be living cells, whereas nuclei exhibiting magenta color, as a consequence of Hoechst and PI colocalization signal, are consider dead cells (b–e). Cytotoxic concentration 50% (CC50; in μg/ml) is defined as the lactoferrin’s concentration required to disrupt the plasma membrane of 50% of the cell population after 24 h of incubation. CC50 was calculated via linear interpolation (https://www.johndcook.com/interpolator.html)

Article Snippet: Briefly, two fluorescence DNA binding dyes were used: Hoechst 33342 (Invitrogen, Eugene, OR), a cell membrane permeable fluorophore labeling living and dead cells, thus, providing with the total number of cells, and propidium iodide (PI; MP Biomedicals, Solon, OH), a dye just staining cells with compromised plasma membrane, specifying the total number of dead cells.

Techniques: Concentration Assay, Incubation, Staining, Positive Control, Standard Deviation, Activity Assay

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Recombinant human lactoferrin induces apoptosis, disruption of F-actin structure and cell cycle arrest with selective cytotoxicity on human triple negative breast cancer cells

doi: 10.1007/s10495-019-01539-7

Figure Lengend Snippet: The rhLf inflicts cell death, DNA fragmentation and morphological changes associated with apoptosis in MDA-MB-231 triple negative breast cancer cells. Cells were exposed to 50 μg/ml of rhLf for 24 h and double stained with Hoechst and PI. Representative live-cell confocal microscope images, captured using Hoechst channel (nuclei; a), brightfield differential interference contrast (DIC; b), propidium iodide channel (c) and overlay of the preceding three images (d), are depicted. The black head arrows are indicating the blebs formation, whereas the redhead arrows are denoting cell shrinkage and chromatin condensation, dead cells, PI positive. Also, the white head arrow is signaling a cell with a non-condensed nucleus, non-cell shrinkage and non-blebs formation, as well as PI negative, therefore a living cell. Also, as above, live-cell images from untreated cells are depicted as follow: e Hoechst channel (nuclei); f brightfield; g propidium iodide channel; and overlay of the preceding three images (h). Scale bar = 10 μm

Article Snippet: Briefly, two fluorescence DNA binding dyes were used: Hoechst 33342 (Invitrogen, Eugene, OR), a cell membrane permeable fluorophore labeling living and dead cells, thus, providing with the total number of cells, and propidium iodide (PI; MP Biomedicals, Solon, OH), a dye just staining cells with compromised plasma membrane, specifying the total number of dead cells.

Techniques: Staining, Microscopy

Journal: Leukemia

Article Title: A novel agent SL-401 induces anti-myeloma activity by targeting plasmacytoid dendritic cells, osteoclastogenesis and cancer stem-like cells

doi: 10.1038/leu.2017.135

Figure Lengend Snippet: Effect of SL-401 on pDCs, MM cells and pDC-induced MM cell growth. (a) pDC (green) and MM (blue) cells were stained with anti-IL-3 Rα-PE antibody or matched isotype control and analyzed by flow cytometry. (Number of patient samples =6). Left panel: A representative histogram shows IL-3Rα/CD123 expression on pDCs (green) and MM cells (blue). Right panel: Frequency of IL-3 Rα/CD123 expression on patient pDCs and tumor cells. The data is presented as mean fluorescence intensity (MFI) of IL-3Rα/CD123 expression on pDCs (green closed circles) and MM cells (blue closed circles) isolated from patient BM samples (n =6). MFI is shown for each cell type (P<0.0101 for pDCs versus MM cells) (b) Schematic of SL-401 construction: SL-401 is a targeted therapy directed to IL-3R consisting of IL-3 fused to a truncated DT payload. The IL-3 domain of SL-401, which replaces the binding domain of DT, targets SL-401 to cells that express the IL-3R. (c) Purified pDCs and MM cells from patients (n =9) BM were treated with indicated concentrations of SL-401 for 48 h, and analyzed for apoptosis (IC50 for pDCs: 0.83 ng/ml =13.67 picomolar) (mean ±s.d., P<0.005; n =4). (d) MM.1 S cells (5 × 104 cells per 200 μl), MM.1 R (5 × 104 cells per 200 μl), and pDCs (1 × 104 cells per 200 μl) were cultured either alone or together (1:5 pDC:MM ratio) for 72 h in the presence or absence of indicated concentrations of SL-401, and DNA synthesis was measured by 3H-TdR uptake (mean ±s.d., n =3). (e) Patient (n = 9) MM cells were cultured with or without autologous pDCs for 72 h in the presence or absence of indicated concentrations of SL-401, and DNA synthesis was measured by 3H-TdR uptake (mean ±s.d. of triplicate cultures; P<0.004 for all samples). (f) Purified indicated cell types from MM patient (n =5) and total PBMCs from normal donors (n =4) were treated with DMSO vehicle or SL-401 for 72 h and analyzed for viability (mean ±s.d. of quadruplicate cultures). Anti-pDC activity of SL-401 served as a positive control.

Article Snippet: 24 , 25 Apoptosis was assessed with Annexin V-FITC/Propidium iodide apoptosis detection kit (BD Biosciences, San Jose, CA, USA), followed by analysis using FACS Canto II.

Techniques: Staining, Flow Cytometry, Expressing, Fluorescence, Isolation, Binding Assay, Purification, Cell Culture, DNA Synthesis, Activity Assay, Positive Control