OLIGOMYCIN TITRATION

PI:
USER:

DATE:
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Experiment/Assay Name:

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PURPOSE: This assay is to optimize the concentration of oligomycin required to inhibit ATP
synthesis completely at complex V (ATP Synthase). Effect of FCCP is also dependent upon the
concentration of oligomycin.

DAY 1
Plate optimum number of cells in cell growth media (100L) into each well of Seahorse cell
culture microplate
After allowing 2.5 - 5.0h to attach the cells, add additional 150L of growth media (final
250L).
Hydrate the assay cartridge in calibrant at 37oC in a non-CO2 incubator overnight.
Create an assay template in XF24 software and include all cell culture details and well/group
labels
Make and sterile filter assay media with appropriate substrates (check pH of media prior to
filtering)
Calculate all dilution volumes for Port additions
GROWTH MEDIA CONDITIONS
CELL CULTURE
CELL INFORMATION
MICROPLATE
Name:
Type:
Lot#:

..

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Serial #:
Tissue of Origin:
FCS:.
.

....
.
Species:
Expiry:

Growth Factors:.

.
..
Passage #:

Other Suppl.:
.
.
XF ASSAY CARTRIDGE
Final Vol.:
Lot#:
Seeding Density:

.
..
Source:
Serial #:
Growth media:

.
..

.
Expiry:
Plating date:

.
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Notes: (Cell counted, dilution etc.)

SAMPLE CELL LAYOUT

2
3
4
5

Un-buffered
Mediaml

ASSAY MEDIA
FCS:
..

A
B
C

40K

40K

40K

40K

40K

40K

40K

40K
D

40K

40K

40K

40K

40K

40K

40K

40K

40K

40K

40K

40K

1.0M Glucose:.
..ml
0.2M Glutamine.
....ml
0.1M Pyruvate ..
..ml
pH:

..

.ml
1.0 M HEPES: ...
..ml

Final Volume: .
mL

DAY 2
Check cells for even distribution in each well note each well & take pictures if necessary
note each well and note that they are OK at a minimum
Remove growth media from the specialized microplate and replace it with assay media at
37oC (Final vol. 450L).
Incubate the microplate at 37oC in a non-CO2 incubator for 1h (Start Time . End
Time...)
Load optimum concentrations of inhibitors into each port of the cartridge
Open assay template on XF24 Software and run the Assay - first calibration using calibrant,
replace utility plate with Seahorse microplate seeded with cells to measure OCR & ECAR
Perform normalization assay (cell count, protein concentration etc.)
Notes:

PREPARATION OF INHIBITORS:
Load each port with each compound 10 Conc. of the desired final Conc. in each well from the
frozen stock of the compound. (See Port loading template on the next page)
For Example: if the final desired concentration of oligomycin in each well is 1.0 g/mL, dilute
from the frozen stock to prepare a concentration of 10g/mL to load into each port.

PORT A - Oligomycin
Port A will be loaded with oligomycin or media (See port loading template next page)
Experimental Wells = 50L of OLIGOMYCIN
Control-Control Wells = 50L of Media
Temperature Control Wells = 50L of Media
Frozen Stock Conc.: 4.0 mg/mL
Port Conc.: 20, 10, 5.0, 2.5 g/mL
Final concentration in the well: 2, 1, 0.5, and 0.25 g/mL
Procedure:
Dilute 10L of frozen oligomycin stock in 1.990 L of assay media (20g/mL)
Dilute 1.0 mL of above (20g/mL) with 1.0 mL of Assay Media (10g/mL)
Dilute 1.0 mL of above (10g/mL) with 1.0 mL of Assay Media (5.0g/mL)
Dilute 1.0 mL of above (5.0g/mL) with 1.0 mL of Assay Media (2.5g/mL)
Load 50L of oligomycin/media to PORT A of corresponding wells (See the template
below).

PORT B - FCCP
Ports labelled with B are loaded with 55L of optimized conc. FCCP/media.
Experimental Wells = 55L of different concentrations of FCCP (see below for template)
Control-Control Wells = 55L of Media
Temperature Control Wells = 55L of Media
Load 55L of optimized concentration of FCCP or media to PORT B

PORT C Rotenone/Antimycin A
Ports labelled C for all the wells are loaded with 60L of 40M ANTI-A/10 M ROTE or media.
Experimental Wells = 60L of Anti-A/Rote (see below for preparation)
Control-Control Wells = 60L of Media
Temperature Control Wells = 60L of Media
Frozen Stock Conc.:
1. Antimycin A: 5.0 mM
2. Rotenone: 1.0 mM
Port Conc.:
1. Antimycin A: 40 M
2. Rotenone: 10 M
Final concentration in the well:
1. Antimycin A: 4.0 M
2. Rotenone: 1.0 M
Procedure:
Dilute 16L of frozen antimycin and 20 L of rotenone stock in 1964 L of assay media
Load 60L of Antimycin/Rotenone or media to PORT C.

PORT D TMPD/Ascorbate
Ports labelled D for all the wells are loaded with 65L of TMPD/Ascorbate or media.

Experimental Wells = 65L of TMPD/Ascorbate (see below for preparation)

Control-Control Wells = 65L of Media
Temperature Control Wells = 65L of TMPD/Ascorbate
Frozen Stock Conc.:
3. TMPD: 200 mM
4. Ascorbate: 800 mM
Port Conc.:
1. TMPD: 5.0 mM
2. Ascorbate: 20 mM
Final concentration in the well:
1. TMPD: 0.5 mM
2. Ascorbate: 2.0 mM
Procedure:
Dilute 45L of frozen Ascorbate and 45 L of TMPD stock in 1710 L of assay media
Load 65L of TMPD/ Ascorbate to PORT D or media.

Port addition Template

A

1
A = Media (50L)
B= Media (55 L)
C = Media (60 L)
D = T/A (65 L)
A = oligo 1g/mL (50
L)
B = Optimum FCCP
(55 L)
C = R/A (60 L)
D = T/A (65 L)

A = oligo 0.5g/mL
(50 L)
B = optimum (55 L)
C = R/A (60 L)
D = T/A (65 L)

A = oligo
0.25g/mL(50L)
B = optimum FCCP
(55 L)
C = R/A (60 L)
D = T/A (65 L)

A = Media
(50L)
B= Media (55
L)
C = Media (60
L)
D = Media (65
L)

INSTRUMENT PROTOCOL:
1. Calibrate
2. Equilibrate
3. Loop 5 times
a. Mix 3min
b. Time delay 2 min
c. Measure 3min
4. Loop end
5. Inject Port A
6. Loop 3 times
a. Mix 3min
b. Time delay 2 min

4
5
6
A = oligo 2g/Ml (50 L)
B = Optimized FCCP (55 L)
C = Rot/Anti (60 L)
D = T/A (65 L
A = Media
A = oligo 1g/mL (50 (50L)
A = oligo 1g/mL
L)
B= Media (55
(50 L)
B = optimum FCCP
L)
B = optimum FCCP
(55 L)
C = Media (60 (55L)
C = R/A (60 L)
L)
C = R/A (60 L)
D = T/A (65 L)
D = T/A (65
D = T/A (65 L)
L)
A = oligo 0.5g/mL (50 L)
A = Media (50L)
B = optimum FCCP (55 L)
B= Media (55 L)
C = R/A (60 L)
C = Media (60 L)
D = T/A (65 L)
D = T/A (65 L)

A = oligo 0.25g/mL (50 L)

B = optimum FCCP (55 L)
C = R/A (60 L)
D = T/A (65 L)

A = Media
(50L)
B= Media (55
L)
C = Media (60
L)
D = T/A (65
L)

c. Measure 3min
7. Loop end
8. Inject Port B
9. Loop 3 times
a. Mix 3min
b. Time delay 2 min
c. Measure 3min
10.Loop end
11.Inject Port C
12.Loop 3 times
a. Mix 3min
b. Time delay 2 min
c. Measure 3min
13.Loop end
14.Inject Port D
15.Loop 3 times
a. Mix 3min
b. Time delay 2 min
c. Measure 3min
16.Loop end
17.Program end