Facs aria machine

A necrosis/apoptosis assay kit (Abcam, Cambridge, UK) was used to detect apoptosis and necrosis levels in H1299 cells according to the manufacturer’s instructions. Seeding of H1299 cells was performed in 6-well plates (250,000 cells/well). After 48 h, PK 11195 at a concentration of 25 µM, was applied for 24 h then followed by exposure of 24 h to 0.5 mM of CoCl₂. Cells were trypsinized using 600 µL trypsin (Trypsin EDTA Solution B (0.25%), EDTA (0.05%)) (Biological industries, Beit Ha’Emek, Israel) centrifuged (660× g for 5 min at room temperature) then the supernatant was discarded, and the cells were resuspended in 200 µL assay buffer (provided in the kit) and transferred to Eppendorf tubes. Staining was conducted by adding 2 µL of Apopoxin to 100 µL of sample for apoptotic cells detection, 1 µL of 7-AAD to 100 µL of sample for necrotic cells detection with subsequent incubation in the dark for 1 h. Before reading the samples, 300 µL of assay buffer were added to each sample. An Aria FACS machine (BD bioscience, San Jose, CA, USA) was used with Ex/Em = 490/525 nm for detection of apoptosis, Ex/Em = 550/650 nm for detection of necrosis. The 10th version of FlowJo (LLC, Ashland, OR, USA) was used for calculation.

For the detection of the levels of apoptosis and necrosis following CS exposure for different time periods, we used the Apoptosis/Necrosis detection kit (Abcam, Cambridge, UK), according to the manufacturer’s instructions. The cells were trypsinized, centrifuged (660× g, 5 mins, 4 °C), and collected. For every sample, 200 µL of assay buffer was added followed by the addition of: 2 μL Apopxin Green Indicator (200X), 1 μL 7-AAD (200X), and 1 μL CytoCalcein violet 450 (200X) to detect apoptosis, necrosis, and intact cells, respectively. After 60 mins of incubation, fluorescence was measured using Aria FACS machine (BD bioscience, San Jose, CA, USA) and the results were calculated by FlowJo (10th version, FlowJo LLC, Ashland, OR, USA).

GFP expressing CRISPR/Cas9 knockout plasmids for mouse Htt (sc-420825) and Fmr1 (sc-420392) or CRISPR/Cas9 control plasmid (sc-418922) were purchased from Santa Cruz Biotechnology. Mouse striatal cells were transfected with knockout plasmids by PolyFect (Qiagen) following recommendations by the company. The transfected cells were harvested 48 h later, washed, and resuspended in the sorting buffer (containing 1× phosphate-buffered saline (Ca/Mg⁺⁺ free), 2.5 mM EDTA, 25 mM HEPES pH 7.0, 1% fetal bovine serum, and 10 unit/ml DNase1). The GFP expressing cells were sorted using a BD biosciences Aria FACs machine. The sorted cells were plated and maintained in DMEM high glucose containing 10% FBS at 33 °C.