Agilent Seahorse XF Plasma Membrane
Permeabilizer (PMP) Quickstart Guide
For use with Agilent Seahorse XFe, XFp, and XF
Analyzers
Product Description 2
Mitochondrial Assay Solution 4
References 6
Product Use and Limitations, Warranty, Disclaimer 7

Kit part number 102504-100

Agilent Technologies

Product Description
The Seahorse XF Plasma Membrane Permeabilizer (PMP) (p/n 102504-100) is
an engineered, recombinant cytolysin protein that has the ability to form poresin cellular plasma membranes via oligomerization. Unlike mostdetergent-based permeabilizers, it targets the cellular plasma membraneselectively, leaving the mitochondrial membrane intact. The permeabilization
process with this product is consistent, reliable and accurate. Therefore there
is no need to perform a concentration titration for every experiment. In
general, 1.0 nM PMP is sufficient for most cell types. These unique features
allow to study mitochondrial function without the need to isolate
mitochondria, resulting significant improvement and simplification of yourworkflow and consistent results for assays.
The utility of this product has been demonstrated in assessing mitochondrial
respiratory complexes, measuring substrate oxidation, and other applications.
For information on these applications, please visit the technical overviews on
Agilent website and/or check the reference list at the end of the document.
http://www.agilent.com/cs/library/technicaloverviews/public/5991-7157EN.pdf
http://www.agilent.com/cs/library/technicaloverviews/public/XF-PMP-Limite
d-Tech-Brief-WEB.pdf
http://www.agilent.com/cs/library/technicaloverviews/public/xf%20pmp%20s
ubstrate%20oxidation.pdf

Contents
One microfuge tube containing 25 µL of 10 µM PMP solution in HNG buffer (50mM Hepes, pH 7.4, 100 mM NaCl, 10% glycerol [v/v]). Each tube contains
enough material for six 24-well or 96-well microplates. The reagent box
contains five slots to store 4 µL aliquots upon arrival, or after the initial thaw.

Storage conditions
Upon arrival, store at -20 °C. The PMP stock reagent has a minimum of
6 months shelf life when properly stored. Storage of diluted PMP reagent is notrecommended. Multiple freeze/thaw cycles are not recommended. Make 4 µL
aliquots into fresh tubes (user supplied) upon arrival, or after the initial thaw.

Materials required (not included)• Agilent Seahorse XF 24/96, XFe 24/96, and XFp Analyzers• Agilent Seahorse XF/XFe/XFp FluxPak or FluxPak mini
• Mitochondrial Assay Solution (MAS)

2Seahorse XF Plasma Membrane Permeabilizer (PMP) Quickstart GuideReagent preparation
It is recommended that PMP be thawed on ice, mixed gently, and diluted
directly into MAS (+ other supplements) immediately prior to the start of theassay.
Table 1

Reagent preparation*Reagent

Source3X MAS

Amount for 1.0 L of 3X MAS

MannitolSigma

660 mM

120.23 g

SucroseSigma

210 mM

71.88 g

KH2PO4Sigma

30 mM

4.08 g

MgCl2Sigma

15 mM

15 mL of 1.0 M solution

HEPES

Sigma

6 mM

6 mL of 1.0 M solution

EGTA

Sigma

3 mM

12 mL of 0.25 M solution

Fatty Acid Free (FAF) BSA

Sigma

0.6% (w/v)

6.0 g* BSA can be omitted from the MAS buffer, but greater concentration of PMP will be required to achieve a similarlevel of permeabilization.

Seahorse XF Plasma Membrane Permeabilizer (PMP) Quickstart Guide3Mitochondrial Assay Solution
To make 1.0 L of 3X MAS
1 Combine the reagents in Table 1 on page 3 in 750 mL dH2O.
2 Bring the volume to 950 mL with dH2O, warm to 37 °C, and ensure allreagents are completely dissolved.
3 Adjust the solution to pH 7.4 with KOH.
4 Add dH2O to bring the final volume to 1.0 L.
5 Sterile filter the solution, and store it at 4 °C.

Use of 3X MAS
Use 3X MAS buffer as a stock buffer to prepare the assay medium. Combine
the 3X MAS with substrates, ADP, PMP, and dH2O to achieve 1X MAS with theappropriate final concentrations of additives. This buffer should also be used
when preparing injections for an PMP experiment.

Day prior to assay
1 Hydrate the XF cartridge.
2 Plate the cells of interest on an XF cell culture microplate.
3 Prepare stock reagents and solutions.

Day of assay
1 Prepare solutions as required.
2 Load the XF cartridge injection ports.
3 Begin cartridge calibration, after cartridge calibration is complete, washthe cells once with 1X MAS, then add the appropriate final volume ofprewarmed (37 °C) assay buffer to the wells.
4 After the final volume addition, insert the microplate into the Seahorse XFAnalyzer. (Note: Unlike standard XF assays, do not incubate at 37 °C in anon-CO2 environment.)

Assay buffer
Agilent Technologies recommends using a nonionic mannitol plus asucrose-based buffer (MAS, composition provided in “To make 1.0 L of 3XMAS”) for permeabilization assays.

4Seahorse XF Plasma Membrane Permeabilizer (PMP) Quickstart GuideAgilent also recommends adding XF PMP + ADP + substrates of interest to theXF assay buffer after the wash step, and not as an injection during the assay.
Permeabilization just before the XF assay limits the amount of time the cell is
exposed to a nonionic buffer and, thus, reduces changes in cell volume andpotential cell adhesion issues.
Table 2

Suggested final concentrations for XF PMP assays

Substrates

AmountADP

4 mM

Succinate/Rotenone

10 mM/2 µM

Pyruvate/Malate

10 mM/0.5 mM

Glutamate/Malate

10 mM/10 mM

Palmitoyl Carnitine/Malate

40 µM/0.5 mM

Optimization of XF PMP assays
XF PMP Concentration
Most cell types (cell lines and primary cells) work well with 1.0 nM final
XF PMP, however optimization of the concentration may be required for somecell types. Agilent Seahorse recommends titrating XF PMP between 0 and
3.0 nM (for example, 0, 0.1, 0.3, 1, 2, and 3 nM) to obtain optimal XF PMP
concentrations.

Strategies for more robust adherence of cells
Agilent recommends using the method discussed above for XF PMP assays,
which limits exposure of the intact cell to a nonionic media, and tends toreduce issues with cell adherence. If cell adhesion remains problematic, try:
• Seeding cells at a lower density, but allowing two days of growth• Using a plate coating• Slightly reducing the concentration of XF PMP

Mix, wait, measure times for XFe/XF instrument commands
Mix, wait, measure times are different than typical XF assays to reduce assay
time and potential for loss of cell adherence upon permeabilization. See
Table 3 on page 6 for recommended mix, wait, measure times for XF PMP
assays.

NOTE

There is no equilibration step in the instrument command protocol.

Seahorse XF Plasma Membrane Permeabilizer (PMP) Quickstart Guide5

Table 3

Recommended mix, wait, and measure times for XF PMP assay 24-well or96-well

CommandTime (minutes)

Mix

0.5

Wait

0.5

Measure2.0

More information on PMP can be found atwww.agilent.com/en-us/products/cell-analysis-(seahorse)/seahorse-xf-consum
ables/kits-reagents-media/seahorse-xf-plasma-membrane-permeabilizer.

References
1 Divakaruni, A. S.; et al. Thiazolidinediones are acute, specific inhibitors ofthe mitochondrial pyruvate carrier. PNAS, 2013, 110(4): 5422-5427.
2 Divakaruni, A.S.; et al. Analysis and interpretation of microplate-based

oxygen consumption and pH data. Methods Enzymol., 2014, 546:309-354
3 Divakaruni, A. S.; et al. Measuring mitochondrial function in Permeabilized

Cells using the Seahorse XF Analyzer or a Clark-Type oxygen electrode.
Current Protocols in Toxicology, 2014, 60: 25.2: 25.2.1 - 25.2.16

6Seahorse XF Plasma Membrane Permeabilizer (PMP) Quickstart GuideProduct Use and Limitations, Warranty, Disclaimer
Agilent Technologies manufactures products for a number of intended uses.
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Seahorse XF Plasma Membrane Permeabilizer (PMP) Quickstart Guide

7

Agilent Technologies

www.agilent.com
For Research Use Only. Not for use in diagnostic procedures.
This information is subject to change without notice.

© Agilent Technologies, Inc. 2017
Published in the USA, March 2017
Revision B*102505-000*
102505-000