Fluo-DHA(NBD荧光标记双氢青蒿素)

Fluo-DHA是一种由NBD基团标记的双氢青蒿素（DHA）荧光探针，主要用于青蒿素药理学研究

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1mg

￥3125.00

2500.00

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5mg

￥8750.00

7000.00

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10mg

￥12500.00

10000.00

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25mg

￥16000.00

0.00

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100mg

￥20000.00

0.00

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Background

Fluo-DHA是一种由NBD基团标记的双氢青蒿素（DHA）荧光探针，主要用于青蒿素药理学研究。¹

Protocol

Quantification of Fluo-DHA uptake was performed using flow cytometry. GIE were preincubated or not with 100 µM NPPB or with 100 µM Furosemide for 30 min and then incubated with 1 µM of Fluo-DHA for 2 h at 37 °C, 5% CO2 and 5% O2. To address the role of cAMP-signaling in Fluo-DHA uptake, early GIE (day 2 post NAG treatment) were preincubated 30 min with 100 µM H89, 10 µM KT5720 or 100 µM 8Br-cAMP, and mature GIE (days 8–11 post NAG treatment) were preincubated 30 min with tadalafil or sildenafil at different concentrations (from 10 to 100 µM). To assess the specificity of Fluo-DHA uptake, early GIE were preincubated or not with 20 µM DHA for 2 h and then incubated with Fluo-DHA (1 µM) for 2 h. DHA alone (20 µM) and HPA-NBD (1 µM) were used as control. Twenty minutes before the end of incubation, GIE were stained with Hoechst 33342 (1/10,000). Cells were then washed with PBS 1× and fixed for 10 min at room temperature with PBS, 1% PFA and 0.25% glutaraldehyde. The percentage of Fluo-DHA-positive cells was quantified using Fortessa (BD Biosciences) cytometer.²

Fluorescence microscopy.To visualize the uptake of Fluo-DHA,GIE wereincubated 2h with Fluo-DHA or dimethyl sulfoxide(DMSO) 0.1% at 37℃. Cells were stained with Hoechst 33342 (1/20,000,Thermo Fisher Scientific) for 10 min at 37°C.After one wash with phosphate-buffered-saline(PBS)1x,cells were fixed for10 min at room temperature with 1% paraformaldehyde(PFA) (Electron Micro-scopy Science) and 0.25% glutaraldehyde in PBS 1x.After three washes with PBS 1x,infected cells were observed on a glass slide at x100 mag-nification using a Leica DMi8 microscope.²

参考文献：

1.Sissoko, A. et al. A chemically stable fluorescent mimic of dihydroartemisinin, artemether, and arteether with conserved bioactivity and specificity shows high pharmacological relevance to the antimalarial drugs. ACS. Infect. Dis. 6, 1532–1547 (2020).DOI:10.1021/acsinfecdis.9b00430

2.Guillaume Bouyer, Daniela Barbieri, Florian Dupuy.et al.Plasmodium falciparum sexual parasites regulate infected erythrocyte permeability.Nature communications biology.726 (2020) .DOI:10.1038/s42003-020-01454-7