488 cytochrome c apoptosis detection kit  (Thermo Fisher)

Journal: PeerJ

Article Title: Palmitic acid induces neurotoxicity and gliatoxicity in SH-SY5Y human neuroblastoma and T98G human glioblastoma cells

doi: 10.7717/peerj.4696

Figure Lengend Snippet: Annexin V-Alexa Fluor® 488/PI flow cytometric analysis of apoptosis and cell cycle in SH-SY5Y and T98G cells after 48 h of PA treatment. SH-SY5Y and T98G cells were treated with increasing concentrations (0, 100, 200 and 300 µM) of PA for 48 h. Cells were stained with annexin V-Alexa Fluor® 488 and PI followed by flow cytometric analysis. Representative dot plots from one experiment are shown. −/+, necrosis; +/+, late apoptosis; −/−, live cells and +/−, early apoptosis. (A) SH-SY5Y. (B) T98G. Cell cycle distribution was also analysed by flow cytometry. Representative DNA content histograms from one experiment were presented. R1, Sub-G-0/G 1 phase; R2, G 0 /G-1 phase; R3, S phase and R4, G 2 /M phase. (C) SH-SY5Y. (D) T98G. (E) Statistical graph of the ‘ +/− quadrant’ of the dot plots of SH-SY5Y and T98G cells. Data represent the mean ± S.E.M. of three independent experiments. * represents p

Article Snippet: The cells were stained with annexin V and PI using Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Cat. No.: V13241; Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Flow Cytometry, Staining, Cytometry

Journal: Scientific Reports

Article Title: Melamine promotes calcium crystal formation in three-dimensional microfluidic device

doi: 10.1038/s41598-018-37191-5

Figure Lengend Snippet: Preformed CaP crystals and melamine (Mel)-induced CaOx crystal formation increases ROS release, LDH release, necrosis and apoptosis. To evaluate the effect of preformed CaP and Mel-induced CaP/CaOx crystal formation on 3D cultured HK2 cells, we measured H 2 O 2 release (unit: relative fluorescence unit; RFU) representing ROS production ( A ); cells undergoing apoptosis, Alexa Fluor-labeled annexin-V staining ( B , C ); RT-PCR blots representing mRNA expression of genes BAX-1 (506 bp) and BCL-2 (147 bp), and GAPDH (336 bp) ( D ). Full RT-PCR blots are provided in the supplementary information. Quantification of BAX/BCL2 ratio ( E ) as apoptosis (normalized to GAPDH as an internal control); PI labeled ( F , G ) representing necrosis and cellular damage LDH release ( H ). Schematic diagram showing proposed mechanism (I) (1) CaP/CaOx/mixed + Mel crystal interactions. (2) Generates ROS formation within the PT cell. (3) ROS production leads to release of H 2 O 2 . (4) ROS induced cell death via apoptosis and necrosis. (5) Cell debris generated from dead cells helps in further aggregation of crystals. Scale bars in B and F, 50 µm.

Article Snippet: Both apoptotic and necrotic cells were assessed by using Alexa Fluro 488 Annexin V/Dead Cell apoptosis Kit (Thermoscientific) as described previously .

Techniques: Cell Culture, Fluorescence, Labeling, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Generated

Journal: eLife

Article Title: Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

doi: 10.7554/eLife.56469

Figure Lengend Snippet: Knockoff development using a model substrate. ( a ) Inhibitor toxicity measured in HEK293T cells using a standard dead cell apoptosis assay. Healthy cells are not fluorescent (green), dead cells are propidium iodide (PI) and annexin V (AV) positive (red). Apoptotic cells are only AV positive (grey), while necrotic cells are only PI positive (black). Hoescht nuclear counterstain was to determine the total number of cells. Values are mean +/- SEM, n = 7–8 for each condition from three independent trials. ( b ) Inhibitor toxicity measured in primary cultures of rat cortical neurons using PI (Dead) and Hoechst (Total) stains. Values are mean +/- SEM, n = 10 fro each condition from two independent trials. ( c ) Representative anti-GFP immunoblots showing time-course of cleavage for DNV and PRV washout with the modified EDVVCC/QMSY model. PRV has the fastest apparent dissociation from the protease, as the model is almost completely cleaved within 1 hr following washout. ( d ) Representative, individual ROI (black) and average (green) iGluSnFR traces showing the Ca 2+ -dependence of release in WT rat hippocampal neurons transduced with iGluSnFR. The examples shown were from a single FOV for each condition. ( e ) Quantification of miniature glutamate transients (mGT) in WT rat hippocampal neurons at 15 DIV using iGluSnFR ( Marvin et al., 2013 ) in 2 mM extracellular Ca 2+ . One µM TTX was used to block action potentials where indicated; in some experiments, forskolin (10 µM) was also included to increase mGT frequency. Addition of PRV drastically increased the rate of spontaneous glutamate release events; TTX blocked this increase. A low dose of PRV (0.5 µM) did not influence spontaneous release. Two separate trials were conducted; bars represent means and individual dots represent events/s from a FOV (n) imaged for one minute, n = 4 to 10. ( f ) Normalized, average traces (n = 5 to 6) of evoked glutamate release from a single action potential in WT neurons, and WT neurons treated with 0.5, 1, or 3 µM PRV, in 1 mM extracellular Ca 2+ . ( g ) Representative confocal images of 20 DIV rat hippocampal neurons stained with anti-PSD95, anti-vGLUT1, and anti-MAP2. Neurons grown in vehicle, 0.5 µM, or 5.0 µM PRV. Quantitation of synapse number to the right, control (grey) 0.5 µM (green), or 5.0 µM PRV (blue).

Article Snippet: Samples were then immediately imaged using widefield microscopy, using standard blue (Hoechst), green (annexin V), and red (PI) filter sets.

Techniques: Apoptosis Assay, Western Blot, Modification, Transduction, Blocking Assay, Staining, Quantitation Assay