Seahorse XF24, Руководство по обучению

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Basic Procedure
Contact Seahorse Technical Support: www.seahorsebio.com/techsupport
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Protocol:
1. Warm the pre-made XF Cell Mito Stress Test Assay Medium to 37
o
C.
Adjust pH to 7.4 ± 0.1 at 37
o
C.
2. Retrieve your cell plate from the CO
2
incubator. Note the time.
3. Look at cells under the microscope to:
a. Confirm cell health, morphology, seeding uniformity and purity (no contamination).
b. Ensure cells are adhered, and no gaps are present.
c. Make sure no cells were plated in the background correction wells.
4. Wash cells with XF Cell Mito Stress Test Assay Medium
a. Remove all but 50 µL of the culture medium from each well.
b. Rinse cells two times with 1 mL of assay medium.
c. Add assay medium to each well for a final volume of 500 μL/well
5. Look at cells under the microscope to ensure that cells were not washed away.
6. Place the plate in a 37°C incubator
without CO
2
for one hour prior to the assay.
7. Prepare Stock Compounds
a. Important: Use compounds the same day they are reconstituted. Do not refreeze. Discard any
remaining compound.
b. Remove foil pouch from XF Cell Mito Stress Test Kit box. Each pouch contains reagents sufficient
for a complete XF Cell Mito Stress Test in a 96 or 24 well XF Cell Culture Microplate.
c. Allow compounds to warm to room temp in the sealed pouch for approximately 15 minutes.
d. Open pouch and remove the three tubes containing oligomycin (blue cap), FCCP (yellow cap),
and rotenone/antimycin A (red cap). Place tubes in a small tube rack.
e. Resuspend contents of each tube with prepared assay medium in volumes described in table
below with a p1000 pipette. Gently pipette up and down (~10 times) to solubilize the
compounds.
Volume of Assay Medium Final Concentration
Oligomycin 630 µL 100 µM
FCCP 720 µL 100 µM
Rotenone / AntimycinA 540 µL 50 µM
8. Prepare your compounds that you will load into the cartridge ports.
a. Prepare 3 mL of oligomycin in assay medium to achieve the desired final concentration
determined in Training experiment 1 (or use 1 µM, which works for most cell types).
Port A
Oligomycin [Final well]
(µM) Stock
volume
(µL) Medium
volume
(µL)
0.5 150 2,850
1.0 300 2,700
2.0 600 2,400