7. Trypsinise the cells, and suspend in appropriate growth media.
8. Mix approximately 10 μL cell suspension in a 1:1 ratio with
0.4% trypan blue and perform cell count.
9. Seed the appropriate number of cells, typically 10,000–20,000
cells in 80 μL of media per well in all but the 4 corner wells.
These are used to determine background measurements and
must remain cell-free (see Notes 3–5). Use a multichannel
pipette to minimise sampling error.
10. Leave the plate undisturbed for 20–30 min in the hood to
assure even cell seeding. If cells are clumped in the middle or
edge of the well, a large amount of variation can be observed in
the assay.
11. Place the 96-well cell culture plate in a CO 2 incubator at 37
C,
5% CO 2 and with 95% humidity and monitor after 5–6 h to
observe cell adherence. Allow the cells to settle overnight.
3.3 Day 2 –
Performing the Mito
Stress Test Assay
1. Remove the probe plate from the non-CO 2 incubator.
2. Replace the water in the probe plate with 200 μL of calibration
buffer and return to 37
C non-CO 2 incubator for 1–4 h (see
Note 6).
3. Warm the required volume of unbuffered basal DMEM media
at 37
C and supplement with glucose (10 mM), glutamine
(2 mM) and sodium pyruvate (1 mM).
4. Adjust the pH to 7.4 Æ 0.05, filter sterilise and warm the assay
media to 37
C.
5. Remove all but 20 μL of the growth media from each well and
wash the cells twice gently with 200 μL of assay media (including the background wells A1, A12, H1 and H12) (see Note 7).
After removing the second 200 μL, add 160 μL of media (total
volume in well is 180 μL) and place cells in the non-CO 2
incubator to equilibrate to atmospheric oxygen.
6. While the cells equilibrate, prepare the injection compounds in
assay media and load them into the ports of the cartridge plate
(Fig. 1) as described in Table 1 (see Notes 8 and 9).
7. Using the XFe96 Wave™ software, create a template file of the
protocol by adding details including the name of cell-type(s),
experimental conditions or pretreatments, the reagent names
and concentrations (see Note 10).
8. The machine prompts the probe plate to be inserted for calibration. Remove the lid before inserting the plate.
9. After calibration is complete, the sensor plate is retained in the
machine and the software prompts the exchange of the utility
plate in the loading tray with the cell culture plate.
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Sugandha Bhatia et al.
8. Mix approximately 10 μL cell suspension in a 1:1 ratio with
0.4% trypan blue and perform cell count.
9. Seed the appropriate number of cells, typically 10,000–20,000
cells in 80 μL of media per well in all but the 4 corner wells.
These are used to determine background measurements and
must remain cell-free (see Notes 3–5). Use a multichannel
pipette to minimise sampling error.
10. Leave the plate undisturbed for 20–30 min in the hood to
assure even cell seeding. If cells are clumped in the middle or
edge of the well, a large amount of variation can be observed in
the assay.
11. Place the 96-well cell culture plate in a CO 2 incubator at 37
C,
5% CO 2 and with 95% humidity and monitor after 5–6 h to
observe cell adherence. Allow the cells to settle overnight.
3.3 Day 2 –
Performing the Mito
Stress Test Assay
1. Remove the probe plate from the non-CO 2 incubator.
2. Replace the water in the probe plate with 200 μL of calibration
buffer and return to 37
C non-CO 2 incubator for 1–4 h (see
Note 6).
3. Warm the required volume of unbuffered basal DMEM media
at 37
C and supplement with glucose (10 mM), glutamine
(2 mM) and sodium pyruvate (1 mM).
4. Adjust the pH to 7.4 Æ 0.05, filter sterilise and warm the assay
media to 37
C.
5. Remove all but 20 μL of the growth media from each well and
wash the cells twice gently with 200 μL of assay media (including the background wells A1, A12, H1 and H12) (see Note 7).
After removing the second 200 μL, add 160 μL of media (total
volume in well is 180 μL) and place cells in the non-CO 2
incubator to equilibrate to atmospheric oxygen.
6. While the cells equilibrate, prepare the injection compounds in
assay media and load them into the ports of the cartridge plate
(Fig. 1) as described in Table 1 (see Notes 8 and 9).
7. Using the XFe96 Wave™ software, create a template file of the
protocol by adding details including the name of cell-type(s),
experimental conditions or pretreatments, the reagent names
and concentrations (see Note 10).
8. The machine prompts the probe plate to be inserted for calibration. Remove the lid before inserting the plate.
9. After calibration is complete, the sensor plate is retained in the
machine and the software prompts the exchange of the utility
plate in the loading tray with the cell culture plate.
332
Sugandha Bhatia et al.