Penta·His Alexa Fluor 488 Conjugate

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	Dilution 1:200

Protocol tips
Dilution 1:200

Publication protocol

Flow cytometry: As a control, a CHO cell line stably expressing human roundabout homolog 4 (Robo4-CHO) was prepared by using the same protocol used to prepare Robo1-CHO cells [32]. For flow cytometry, we dissociated 3 × 105 Robo1-CHO and Robo4-CHO cells using 2.5 g/L trypsin containing 1 mM EDTA (Nacalai Tesque). Cells were washed with PBS containing 5% fetal bovine serum (Gibco); subsequent procedures were performed in this buffer. The cells were suspended in 2 nM of B-STag or buffer only, kept on ice for 30 min, washed twice, and suspended in 2 nM of E-SCat or buffer only. The cells were incubated on ice for 2 h, washed twice, suspended in Penta·His Alexa Fluor 488 conjugate (dilution, 1:200; Qiagen), and incubated on ice for 30 min. The cells were washed twice, filtered over a 40-μm cell strainer (Falcon, Corning), and analyzed in a flow cytometer (LSRFortessa, BD Biosciences) equipped with a high-throughput sampler using FACS Sheath Solution (BD Biosciences). Light scattering and fluorescence data were collected for 10,000 cells per condition. For fluorescence detection, a 488-nm-wavelength blue laser was used for excitation; emission was detected by using a standard filter at 530/30 nm. Data were analyzed by using FlowJo (version 10). Cell distribution plots were generated using a biexponential x-axis of the area of AlexaFluor488.

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