Cell Meter™ Fluorimetric Intracellular pH Assay Kit

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Overview SDS Protocol

Platform

Fluorescence microplate reader

Excitation	490 nm
Emission	535 nm
Cutoff	515 nm
Recommended plate	Black well/Clear bottom

Other instruments

ArrayScan, FDSS, FLIPR, FlexStation, IN Cell Analyzer, NOVOStar, ViewLux

Components

Example protocol

AT A GLANCE

Protocol Summary

for Standard Cell Load (One Plate)

Prepare cells in growth medium
Add equal volume of RatioWorks™ BCFL, AM dye-working solution
Incubate at 37 °C for 1 hour
Read Fluorescence at Ex/Em= 490/535 nm (Cutoff = 515 nm) with 50 µL/well compound addition (or 500/530 nm and 440/530 nm for ratio)

Protocol Summary

for Acid-Load (One 96-well Plate)

Prepare cells in growth medium
Remove the growth medium
Add RatioWorks™ BCFL, AM dye-working solution (50 µL/well/96-well plate)
Incubate at 37°C for 1 hour
Add 220 mM NH₄Cl (5 µL/well)
Incubate at RT for 15 minutes
Read Fluorescence at Ex/Em= 490/535 nm (Cutoff = 515 nm) with 200 µL/well compound addition (or 500/530 nm and 440/530 nm for ratio)

Important Thaw all the kit components at room temperature before starting the experiment.

CELL PREPARATION

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

RatioWorks™ BCFL, AM stock solution

Add 200 µL DMSO into the vial of RatioWorks™ BCFL, AM (Component A) and mix well to make RatioWorks™ BCFL, AM stock solution.
Note 20 µL of reconstituted RatioWorks™ BCFL, AM stock solution is enough for 1 plate of Standard Cell Load. 10 µL of reconstituted RatioWorks™ BCFL, AM stock solution is enough for 1 plate of Acid Load. RatioWorks™ BCFL, AM stock solution can be stored at ≤ -20 ^oC for one month if the tubes are sealed tightly. Protect from light.

PREPARATION OF WORKING SOLUTION

1. For Standard Cell Load (One Plate)

Add 1 mL of 10X Pluronic F127 Plus (Component B) into 9 mL of HHBS (Component C) and mix well to make 1X assay buffer.
Note For cells that require probenecid for loading (e.g. CHO cells), dilute 50 mM ReadiUse™ Probenecid (Component D) at concentration of 1 to 5 mM (prefer 5 mM for CHO cells).
Add 20 µL of reconstituted RatioWorks™ BCFL, AM stock solution into 10 mL of 1X assay buffer and mix well to make RatioWorks™ BCFL, AM dye-working solution. This RatioWorks™ BCFL, AM dye-working solution is stable for at least 2 hours at room temperature.

2. For Acid-Load (One 96-well Plate)

Add 1 mL of 10X Pluronic F127 Plus (Component B) into 4 mL of HHBS (Component C) and mix well to make 1X assay buffer.
Note For cells that require probenecid for loading (e.g. CHO cells), dilute 50 mM ReadiUse™ Probenecid (Component D) at concentration of 0.5 to 2.5 mM (prefer 2.5 mM for CHO cells).
Add 10 µL of reconstituted RatioWorks™ BCFL, AM stock solution into 5 mL of 1X assay buffer and mix well to make RatioWorks™ BCFL, AM dye-working solution. This RatioWorks™ BCFL, AM dye-working solution is stable for at least 2 hours at room temperature.

SAMPLE EXPERIMENTAL PROTOCOL

Run pH Assay for Standard Cell Load

Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) RatioWorks™ BCFL, AM dye-working solution into the cell plate.
Note It is important to replace the growth medium with HHBS buffer (100 µL/well for 96-well plate or 25 µL/well for 384-well plate before dye-loading) if your compounds interfere with the serum.
Incubate the dye-loading plate in cell incubator for 30 minutes, and then incubate the plate at room temperature for another 30 minutes.
Note If the assay requires 37°C, perform the experiment immediately without further room temperature incubation.
Prepare the compound plates by using HHBS or your desired buffer.
Run the pH assay by monitoring the fluorescence at Ex/Em = 490/535 nm (Cutoff = 515 nm) or 500/530 nm and 440/530 nm (Cutoff = 515 nm) for ratio measurements. The compound addition is 50 µL/well (96-well plate) or 25 µL/well (384-well plate).
Note The assay should be complete within 3 to 5 min after compound addition, however a minimum of 8 min data collection are recommended for during assay development.

Run pH Assay for Acid-Load

Remove the growth medium from the cell plate.
Add 50 µL/well/96-well plate RatioWorks™ BCFL, AM dye-working solution into the cell plate.
Incubate the dye-loading plate in cell incubator for 30 minutes, and then incubate the plate at room temperature for another 30 minutes. Note: If the assay requires 37°C, perform the experiment immediately without further room temperature incubation.
Add 5 µL of 220 mM NH₄Cl and centrifuge the plates for 5 seconds. Incubate 15 minutes at room temperature.
Note NH₄Cl solution should be prepared freshly in HHBS (Component C).
Prepare the compound plates by using HHBS or your desired buffer.
Run the pH assay by monitoring the fluorescence at Ex/Em = 490/535 nm (Cutoff = 515 nm) or 500/530 nm and 440/530 nm (Cutoff = 515 nm) for ratio measurements. The compound addition is 200 µL/well/96-well plate.
Note The assay should be complete within 3 to 5 min after compound addition, however a minimum of 8 min data collection are recommended for during assay development.

Images

Figure 1. Carbachol dose response in CHO-M1 cell. CHO-M1 cells were seeded overnight in 60,000 cells per 100 µL per well in a 96- well black wall/clear bottom costar plate. The growth medium was replaced with 50 µL/well of RatioWorks™ BCFL, AM dye-loading solution for 37°C for 1 hour, follow by 15 minutes incubation with 5 µL/well of 220 mM NH4Cl. Carbachol (200µL/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations. The fluorescent signal was generated using Ex/Em = 490/535 nm (Cutoff = 515 nm).

Figure 2. Effects of empagliflozin on Na+/H+ exchanger (NHE) and downstream signaling. Averaged data of the intracellular pH in the control cells and empagliflozin (1 μmol/L)-treated atrial fibroblasts cotreated with or without cariporide (10 μmol/L) for 48 h (n = 6 experiments, statistic test by one-way repeated measures ANOVA). Source: Graph from Empagliflozin suppressed cardiac fibrogenesis through sodium-hydrogen exchanger inhibition and modulation of the calcium homeostasis by Chung et al., Cardiovascular Diabetology, Feb. 2023.

Citations

View all 2 citations: Citation Explorer

Empagliflozin suppressed cardiac fibrogenesis through sodium-hydrogen exchanger inhibition and modulation of the calcium homeostasis
Authors: Chung, Cheng-Chih and Lin, Yung-Kuo and Chen, Yao-Chang and Kao, Yu-Hsun and Yeh, Yung-Hsin and Trang, Nguyen Ngoc and Chen, Yi-Jen
Journal: Cardiovascular Diabetology (2023): 1--15

The Redox Status of Cancer Cells Supports Mechanisms behind the Warburg Effect
Authors: Moreira, Jorgelindo Da Veiga and Hamraz, Minoo and Abolhassani, Mohammad and Bigan, Erwan and Pérès, Sabine and Paulevé, Loic and Nogueira, Marcel Levy and Steyaert, Jean-Marc and Schwartz, Laurent
Journal: Metabolites (2016): 33

References

View all 34 references: Citation Explorer

Determination of intracellular pH using sensitive, clickable fluorescent probes
Authors: Yapici NB, M and alapu SR, Chew TL, Khuon S, Bi L.
Journal: Bioorg Med Chem Lett (2012): 2440

A two-step pH-dependent liquid-liquid extraction combined with HPLC-fluorescence method for the determination of 10-hydroxycamptothecin in mouse liver tissue
Authors: Zheng J, Guo H, Guo N, Ma W, Jing L, Zhang R, Dai Z, Yan X, Wang Y, Wang Z.
Journal: Pharm Biol (2012): 954

Aqueous synthesis of CdTe/CdSe core/shell quantum dots as pH-sensitive fluorescence probe for the determination of ascorbic acid
Authors: Yang SS, Ren CL, Zhang ZY, Hao JJ, Hu Q, Chen XG.
Journal: J Fluoresc (2011): 1123

Flow injection small-volume fiber-optic pH sensor based on evanescent wave excitation and fluorescence determination
Authors: Xiong Y, Huang Y, Ye Z, Guan Y.
Journal: J Fluoresc (2011): 1137

Isoelectric point determination for Glossoscolex paulistus extracellular hemoglobin: oligomeric stability in acidic pH and relevance to protein-surfactant interactions
Authors: Santiago PS, Carvalho FA, Domingues MM, Carvalho JW, Santos NC, Tabak M.
Journal: Langmuir (2010): 9794

Determination of intraliposomal pH and its effect on membrane partitioning and passive loading of a hydrophobic camptothecin, DB-67
Authors: Joguparthi V, Feng S, Anderson BD.
Journal: Int J Pharm (2008): 17

Cadmium telluride quantum dots as pH-sensitive probes for tiopronin determination
Authors: Wang YQ, Ye C, Zhu ZH, Hu YZ.
Journal: Anal Chim Acta (2008): 50

Simultaneous determination of pH, urea, acetylcholine and heavy metals using array-based enzymatic optical biosensor
Authors: Tsai HC, Doong RA.
Journal: Biosens Bioelectron (2005): 1796

Determination of the pH of the Cryptococcus neoformans vacuole
Authors: Harrison TS, Chen J, Simons E, Levitz SM.
Journal: Med Mycol (2002): 329

Comment on "Determination of electrophoretic mobilities and hydrodynamic radii of three humic substances as a function of pH and ionic strength"
Authors: Schmitt-Kopplin P., undefined
Journal: Environ Sci Technol (2002): 3041