Sh 800z sorting flow cytometer

After visualization of optimal transfection conditions, transfection efficiency was quantified by measuring EGFP expression using flow cytometry. Transfected HEK 293T cells were released by trypsinization, centrifuged, and resuspended in DPBS. Flow cytometry was performed using a Sony SH-800Z sorting flow cytometer (Sony Biotechnology, San Jose, CA, USA) equipped with 405, 488, 561, and 638 nm lasers capable of detecting up to 8 parameters (6 fluorescent and 2 scatter channels). Sony cell sorter software was used to operate the instrument. Untransfected HEK 293T cells were used as a negative control. The 488 nm excitation laser was used with a 100 µM chip operating at 6 psi. EGFP (or EGFP–ApAFP752) fluorescence was detected using the fluorescence 2 (FL2) emission detector at 525 nm. The percentage of HEK 293T cells expressing EGFP (or EGFP–ApAFP752) was measured by gating for a region with <1% of untransfected HEK 293T cells in the EGFP gated region, as described previously [42 (link)]. The gating strategy can be found in Figure S2. All flow cytometry data were analyzed using FlowJo software (Becton Dickinson, Franklin Lakes, NJ, USA).

Enriched CD24-positive cells (~10⁷) were simultaneously stained with Vybrant™ FLICA pan-Caspase Apoptosis Assay Kits for flow cytometry (Invitrogen/Thermo Scientific, Waltham, MA, USA) 1:150 and APC conjugated anti-human Ganglioside GD2 Antibody (BioLegend, San Diego, CA, USA) 1:100 in 0.25 mL of IsoFlow Sheath Fluid (Beckman Coulter, Pasadena, CA, USA) for 30 min at 37 °C. Then the cells were washed once with warm IsoFlow fluid and measured by a Sony SH800Z Sorting Flow Cytometer (Sony Biotechnology, Tokyo, Japan) with automatic channel compensation.