1x1 image pixel for data collection
Intracellular pH Calibration Buffer Kit
Intracellular pH Calibration Buffer Kit
Invitrogen™

Intracellular pH Calibration Buffer Kit

The Intracellular pH Calibration Buffer Kit enables the quantitation of intracellular pH when used in conjunction with pHrodo™ Red AM,Read more
Have Questions?
Catalog NumberQuantity
P353791 kit
Catalog number P35379
Price (USD)
313.65
Online Exclusive
327.00
Save 13.35 (4%)
Each
In stock
Add to cart
Quantity:
1 kit
Price (USD)
313.65
Online Exclusive
327.00
Save 13.35 (4%)
Each
Add to cart
The Intracellular pH Calibration Buffer Kit enables the quantitation of intracellular pH when used in conjunction with pHrodo™ Red AM, pHrodo™ Green AM, or BCECF intracellular pH indicators. pHrodo™ Red AM and pHrodo™ Green AM are weakly fluorescent at neutral pH but increasingly fluorescent as the pH drops. Modification of pHrodo™ red and green dyes with AM ester groups results in an uncharged molecule that can permeate cell membranes. Once inside the cell, the lipophilic blocking groups are cleaved by nonspecific esterases, resulting in a compound that is retained within the intracellular space. Fluorescence intensity of the probes is then an indicator of intracellular pH. Subsequent use of the Intracellular pH Calibration Buffer Kit allows this intracellular pH to be quantified.

The Intracellular pH Calibration Buffer Kit contains a range of pH calibration buffers (pH 4.5, 5.5, 6.5, and 7.5), as well as valinomycin and nigericin, which help equilibrate the pH inside and outside of cells. After using pHrodo™ Red AM or pHrodo™ Green AM and measuring fluorescence intensity under experimental conditions, the cells are then resuspended in one of the calibration buffers to which valinomycin and nigericin have been added, and the fluorescence intensity measured again. This is repeated with the remaining calibration buffers so that a standard curve can be created and used to pinpoint the intracellular pH associated with the experimental conditions.
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Detection MethodFluorescence
FormatCell Culture Plate (Multi-Well), Cell Culture Dish
Quantity1 kit
Sub Cellular LocalizationCytosol
For Use With (Equipment)Fluorescence Microscope, Flow Cytometer, Fluorometer, High Content Analysis Instrument
Product LinepHrodo
Product TypepH Calibration Buffer Kit
Unit SizeEach
Contents & Storage
Store at 2°C to 8°C.

Frequently asked questions (FAQs)

What are the fluorescence excitation/emission maxima for the pHrodo Red AM and pHrodo Green AM, provided in the Intracellular pH Calibration Buffer Kit?

pHrodo Red AM: 560/580 nm
pHrodo Green AM: 509/533 nm

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Why don't I see a significant change in signal for my live-cell fluorescent indicator dye?

Regardless of the type of live-cell indicator dye (e.g., calcium indicators, pH indicator, metal ion indicators), make sure there is no serum during the loading step, which can prematurely cleave dyes with AM esters and bind dyes non-specifically. Always optimize the dye concentration and staining time with a positive control before you run your test samples, to give the best signal-to-background. Always run a positive control with a buffer containing free ions of known concentration and an ionophore to open pores to those ions (for instance, for calcium indicators like Fluo-4 AM, this would include a buffer with added calcium combined with calcimycin, or for pH indicators, buffers of different pHs combined with nigericin). Reactive oxygen indicators, such as CellROX Green or H2DCFDA would require a cellular reactive oxygen species (ROS) stimulant as a positive control, such as menadione. Finally, make sure your imaging system has a sensitive detector. Plate readers, for instance, have much lower detector efficiency over background, compared to microscopy or flow cytometry.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Documents & Downloads

Certificates

Safety Data Sheets

Share catalog number, name or link

1x1 image pixel for data collection