Potassium efflux was confirmed by investigating the intracellular K+ content of the cells using the cell permeable, fluorophore PBFI-AM (Abcam, Cambridge, United Kingdom). After 48 h infection or treatment with the porphyrin extract, supernatant was removed and the cells were washed with Hanks balanced salt solution (HBSS; Gibco). 400 μL loading buffer (PowerLoad, Invitrogen) was added to the wells and 10 μM PBFI-AM and 10 μM Pluronic F-127 (Invitrogen) were included per well, after which the plate was incubated, protected from light, for 2h at 37°C. Afterwards, the loading buffer was removed and the cells were washed with HBSS. Imaging was accomplished using the EVOS FL Auto Imaging System equipped with a 10x objective (excitation: 357 nm, emission: 447 nm, final magnification: 300x). Fluorescence intensity was measured using the ImageJ software.
Pbfi am
Manufactured by Abcam
Sourced in China
PBFI-AM is a fluorescent indicator used for detecting and measuring calcium levels in live cells. It binds to calcium ions and emits a fluorescent signal that can be detected and analyzed.
Automatically generated - may contain errors
Lab products found in correlation
Pluronic f 127, by Thermo Fisher Scientific (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Powerload, by Thermo Fisher Scientific (1 mentions)
Pbfi am, by Thermo Fisher Scientific (1 mentions)
Facsaria sorp, by BD (1 mentions)
Pluronic f 127, by Solarbio (1 mentions)
Sbfi am, by Abcam (1 mentions)
3 protocols using pbfi am
1
Intracellular Potassium Efflux Monitoring
2
Potassium Flux Monitoring by PBFI-AM
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The fluorescent potassium binding probe, PBFI-AM (Abcam) was freshly prepared by mixing with equal volumes of a 25 % (w/v) Pluronic F-127 (Invitrogen) in DMSO. At the endpoint of the cell treatment, cells were loaded with 5 μM PBFI-AM in 1× HBSS for 30 min. Then cells were collected and proceeded to 10 μg/mL propidium iodine (PI) staining for 5 min. Five thousand cells in each sample were analysed by BD FACSAria SORP (BD). The dye of PBFI-AM was activated by the UV and detected by BUV496 channel; the PI was detected by PE-Texas Red channel.
3
Intracellular Ion Measurement in BV-2 Cells
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This procedure was routinely performed as previously reported (41) . The BV-2 cells were seeded into 96well plates to adhere overnight. After treatment with LPS and indicated antagonists, the cells were loaded with either potassium-or sodium-sensitive uorescent dyes, PBFI-AM (5 µmol/L, Abcam) or SBFI-AM (5 µmol/L, Abcam), which were freshly prepared by combining with equal volume of 25% Pluronic F-127 (Solarbio, Beijing, China) for 40 minutes at 37℃. The change in 340 nm/380 nm ratio that represents the alteration of intracellular K + or Na + was analyzed with the multiscan spectrum. Stimulating drugs including ATP and diazoxide were added 30 seconds after the rst measurement.
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