Tali apoptosis kit annexin 5 alexa fluor 488

Cell lines, PBMC, and myeloid/lymphoblastic cell lines were analyzed 30 min and 24 hours after Ultrasound treatment (US), by Flow cytometry Annexin V Alexa Fluor 488 and Propidium Iodide (PI) assay using the Tali® Apoptosis Kit - Annexin V Alexa Fluor® 488 (Molecular Probes, Life Technologies) as recommended by the manufacturer. For Eryptosis assay, RBCs untreated and treated were resuspended in 200 μl of 1X Annexin Buffer plus 10 μl Annexin-V Alexa Fluor, then spun down at 800 x g 10 min washed with PBS + 0.1 % BSA and analyzed by Flow cytometry.

The evaluation of apoptosis and necrosis was conducted in the important treatment groups by Tali Apoptosis kit- Annexin V Alexa Fluor 488^™ (Thermo Fisher Scientific, USA). The whole experiment was done as per the provided kit manual. The samples were analyzed in a flow cytometer (Beckman Coulter, FC500, Brea, CA) using forward scatter and side scatter with a flow rate of the machine maintained set on 30 µl/s.

All reagents were purchased from Sigma Aldrich unless otherwise noted. Cell culture media was obtained from ThermoFisher Scientific and Lipidure®-CM from AMS Biotechnology (Europe) Ltd. The enzymes used for cell isolation were collagenase Type F, collagenase Type 3 (Worthington, NJ, USA), papain (Worthington) and hyaluronidase. Cell culture dishes with glass coverslip bases (Ibidi µ-Dish 35 mm, high) were purchased from Thistle Scientific (UK). Tali™ Apoptosis Kit (Annexin V Alexa Fluor™ 488) and Dil-conjugated oxLDL from Human Plasma (Dil-OxLDL) were both from Invitrogen™ (ThermoFisher Scientific). The antibodies used for immunocytochemistry were mouse anti-SMA-Cy3 (C6198, Sigma-Aldrich), rabbit anti-Galectin 3 (PA579595 ThermoFisher Scientific) and goat anti-rabbit-AlexaFluor633 (A21071, ThermoFisher Scientific), along with Hoechst33342 Solution (ThermoFisher Scientific).
The buffers used during cell isolation were: Mops buffer (145mM sodium chloride, 2mM MOPS, 4.7mM potassium chloride, 1.2 mM monosodium phosphate, 5mM glucose, 0.02 mM EDTA, 2mM sodium pyruvate, 1.2mM magnesium chloride, 2mM calcium chloride, pH 7.4) and isolation buffer (80mM sodium glutamate, 55mM sodium chloride, 6mM potassium chloride, 10mM glucose, 10mM Hepes,1mM magnesium chloride, 0.1mM calcium chloride, 0.2mM EDTA, pH 7.4), with or without 2 mg/ml fatty acid free bovine serum albumin (BSA).

UVA-induced apoptosis in HGFs was detected by a Tali Apoptosis Kit - Annexin V Alexa Fluor 488 and propidium iodide (PI) (A10788, Thermo Fisher Scientific, Tokyo, Japan) according to the manufacture’s protocol. After being washed by PBS (-) twice, cells (5 × 10⁵ to 1 × 10⁶ cells/mL) were stained with Annexin V solution for 20 min in the dark. Cells were then stained with PI solution for 1–5 min. The intensity of the green and red fluorescence was measured with the Tali™ Image Cytometer (Thermo Fisher Scientific, Tokyo, Japan).

Analyses of cell viability, necrosis, and apoptosis were performed using the Tali™ Apoptosis Kit - Annexin V AlexaFluor^® 488 and Propidium Iodide (Invitrogen). The cells were previously treated with the biogenic nanoparticles at a concentration of 0.93 × 10¹² NPs/mL for 24 h, and samples were prepared according to the kit specifications. The reading was performed with the Tali™ Image-Based Cytometer.
The oxidative stress analyses were performed using CellRox Orange^TM reagent (Invitrogen). The cells were previously treated with the biogenic nanoparticles at a concentration of 0.93 × 10¹² NPs/mL for 1 h, and samples were prepared according to the kit specifications. The reading was performed with the Tali™ Image-Based Cytometer. Imaging cytometry assays with each one of the cell lines were performed thrice.