Materials and Reagents

CELLSTAR^® Tissue Culture Plates, 96-well (Greiner Bio One International, catalog number: 655180 )
Sterile racked pipette tips (1 ml and 200 μl) (VWR, catalog numbers: 613-0738 ; 613-0742 )
Sterile basins (Corning, Costar^®, catalog number: 4870 )
Sterile reagent tubes (15 and 50 ml) (VWR, catalog numbers: 89039-668 ; 89039-662 )
Sterile Serological pipettes (5, 10, 25, 50 ml) (Fisher Scientific, catalog numbers: 13-678-11 , 13-678-11D , 13-678-11E , 13-678-11F )
Glass bottles (500 ml) (Fisher Scientific, catalog number: FB8001000 )
HCT-116, SK-Mel-5 and SK-Mel-28 cells (ATCC, catalog numbers: CCL-247 , HTB-70 , HTB-72 )
Seahorse XF24 FluxPak (including sensor cartridges, tissue culture plates, calibrant solution and calibration plates) (Agilent Technologies, Santa Clara, CA)
Trypsin/EDTA (0.25%/2.21 mM) (Corning, catalog number: 25-053-Cl )
1x Ca²⁺/Mg²⁺-free DPBS (Thermo Fisher Scientific, Gibco^TM, catalog number: 14190250 )
Liquid Dulbecco’s modified Eagle’s medium (DMEM) (Corning, catalog number: 10-013 )
Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11150 )
Penicillin/streptomycin solution 100x (Corning, catalog number: 30-002-Cl )
Powder Dulbecco’s modified Eagle’s medium (DMEM) without Na₂HCO₃, without HEPES (Corning, catalog number: 50-013 )
Sodium hydroxide (NaOH) (VWR, catalog number: 97064-476 )
Oligomycin (Merck, catalog number: 495455-10MG )
DMSO (VWR, catalog number: BDH1115-1LP )
FCCP (Cayman Chemical, catalog number: 15218 )
Rotenone (Cayman Chemical, catalog number: 13995 )
Antimycin A (Enzo Life Sciences, catalog number: ALX-380-075-M005 )
Culture media (10% (v/v) FBS) (see Recipes)
Assay media (see Recipes)
NaOH (1 M) (see Recipes)
Oligomycin (10 µM) (see Recipes)
FCCP (5 µM) (see Recipes)
Rotenone (5 µM)/antimycin A (5 µM) (see Recipes)

Equipment

Hemacytometer (Hausser Scientific, catalog number: 1490 )
Biological Safety Cabinet Class II, Type A2 (NuAire, model: NU-425-400ES )
Seahorse XF Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA)
Pipet-Lite Pipette XLS STD 20 XLS (Mettler Toledo, Rainin, model: SL-2XLS+ )
Pipet-Lite Pipette XLS STD 200 (Mettler Toledo, Rainin, model: SL-200XLS+ )
Pipet-Lite Pipette XLS 1000 (Mettler Toledo, Rainin, model: SL-1000XLS+ )
Multichannel Pipet-Lite Pipette XLS 8-CH 1200 (Mettler Toledo, Rainin, model: L8-1200XLS+ )
Multichannel Pipet-Lite Pipette XLS 8-CH 200 (Mettler Toledo, Rainin, model: L8-200XLS+ )
Aspirator pump
Humidified non-CO₂ incubator (XF Prep Station; Agilent Technologies, Santa Clara, CA)
Shallow water bath (Thermo Fisher Scientific, Thermo Scientific^TM, model: Precision 180 )
Pipette controller (BrandTech Scientific, model: Accu-Jet^® Pro , catalog number: 26330)
Humidified, 37 °C, 5% CO₂ incubator (Eppendorf, model: Galaxy^® 170 R )
-20 °C biomedical freezer (Sanyo, model: MDF-U731M )
Autoclave (Consolidated Sterilizer Systems, model: SSR-3A , ADVPB)
Inverted light microscope (Nikon Instruments, model: Eclipse TS100 )
pH-meter with semi-micro electrode (Thermo Fisher Scientific, Thermo Scientific^TM, model: Orion Star^TM A211 , with ROSS 8103BN electrode: (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 8103BN )

Software

Seahorse Bioscience XF24 software
Excel (Microsoft)
GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA)

Procedure

Optimization of seeding density
In an initial experiment, the optimal seeding density is required for each cell type. Typically, the cell density ranges from 10,000 to 60,000 cells per well and can vary widely among cell lines. A first point of orientation can be the cell number that gives confluency of approx. 95% overnight in a 96-well cell culture plate as the seeding surface is comparable to the seahorse culture plate. The seeding number should give a confluent and healthy and consistent monolayer on the day of the assay.
1. HCT-116, SK-Mel-5 and SK-Mel-28 cells were seeded in a Seahorse XF24 cell culture plate at various concentrations ranging from 10,000 to 30,000 cells/well with a two-step seeding technique as described below in Procedure B (Figure 2). Seeding cells in triplicates is recommended.
  
  Figure 2. Plate layout for cell density evaluation. Shown here is the exemplary seeding layout for the SK-Mel-5 and SK-Mel-28 cell lines (seeding density for HCT-116 cells was evaluated on a second plate; not shown).
2. Cells were then assayed in the XF24 instrument as described in Procedure E (without loading compounds into ports) using Table 1 commands.
  
  Table 1. Protocol commands for cell density evaluation
  
  As seen in Figure 3, a linear increase of OCR values with increasing cell density was observed in all three cell lines. ECAR values begin to level off at 20,000 cells/well for SK-Mel-28 and SK-Mel-5 while being much lower and steadily increasing for HCT-116. Thus, a seeding number of 20,000 cells/well for SK-Mel-28 and SK-Mel-5 and of 35,000 cells/well for HCT-116 were chosen to ensure being within the linear response range while having high reading values to observe increases as well as decreases in OCR and ECAR.
  
  Figure 3. Optimization of assay conditions: evaluation of OCR and ECAR depending on the seeding density of three different cell lines
Seeding cells into Seahorse XF24 tissue culture plate (Day 1)
Note: The seeding and growing of cells are performed with good sterile cell culture technique. A two-step seeding method is used to obtain a consistent even monolayer which is vital to obtain consistent and accurate data:
1. Pre-warm culture media, trypsin solution and DPBS to 37 °C.
2. For adherent cells, wash cells with DPBS, and add trypsin and wait until cells begin to detach. Add culture media with serum to deactivate trypsin and pipette up and down to create a uniform cell suspension. Count cells with a hemocytometer and resuspend cells in growth media to the desired final concentration to seed in 100 µl.
3. Plate 100 µl cell suspension into a Seahorse XF24 tissue culture plate. Put media only (no cells) in the background correction wells (A1, B4, C3, D6).
4. Let the culture plate sit for 1 h in the bio-hood without moving it around (in order to let cells settle evenly).
5. Place the culture plate into an incubator (37 °C, 5% CO₂) for 4 h.
6. Carefully add 150 µl growth media (final volume in well 250 µl). Hold the pipette tip at an angle and add to the well side to not destroy even layer of newly attached cells.
7. Let cells grow overnight at 37 °C, 5% CO₂.

Note: The following steps are performed without sterile technique, but caution to keep the cells and equipment as clean as possible.

Hydrate sensors (Day 1)
1. Open XF 24 FluxPak and take out the sensor cartridge (green) and calibration plate (clear) (Figure 4).
  
  Figure 4. Seahorse XF 24 sensor cartridge. A. The sensor cartridge sitting on top of a calibration plate with injection ports shown. B. Bottom side of the sensor plate which shows sensors with embedded fluorophores.
2. Place the sensor cartridge (sensors up) next to the calibration plate (be careful not to touch sensors).
3. Fill each well of the calibration plate with 1 ml of Seahorse XF Calibrant.
4. Lower the sensor cartridge onto the calibrant plate submerging the sensors in calibrant (be careful not to touch walls with sensors).
5. Place in a non-CO₂ 37 °C incubator overnight. To prevent evaporation of the XF Calibrant, verify that the incubator is properly humidified.
Stabilization of instrument (Day 1)
1. Turn on an XF24 Analyzer, open Seahorse Bioscience software and log in.
2. Write the assay template. When planning and writing the assay protocol be careful not to create a protocol that is longer than cells can manage without CO₂ in unbuffered media. Depending on cell type this is 2-3 h. If in doubt, a cell viability assay can be performed after the seahorse assay.
3. Leave the XF24 Analyzer on overnight with XF24 software running and logged in to ensure equilibration to 37 °C.
Seahorse assay (Day 2)
1. Check on the confluency of cells. Evenly spacing of cells is needed, without large cell clumps or blank patches, as this could impair the accuracy of data.
2. Pre-warm assay media to 37 °C.
3. Pre-warm compounds and adjust to pH 7.4 with NaOH (1 M) if necessary.
4. Perform media exchange in a Seahorse XF24 tissue culture plate:
  1. Remove 150 µl growth media with a multichannel pipet.
  2. Add 1 ml assay media with a multichannel pipette.
  3. Remove 1 ml with a multichannel pipette.
  4. Add 475 µl assay media with a multichannel pipette (575 µl final volume).
  5. Place the cell plate into a CO₂-free incubator for approx. 60 min.
5. Load cartridge with desired compounds:
  1. Pre-warm compounds to 37 °C.
  2. Load 50-100 µl of compound into appropriate port of cartridge (for mitochondrial stress test: 64 µl into port A, 71 µl port B, 79 µl port C, 88 µl port D). (see Note 1) Load equivalent amounts of assay media into equivalent port for background wells (see Note 2).
  3. Place back into the incubator (non-CO₂) for 10 min to allow heating up to 37 °C again. Handle carefully, carry only by holding onto the calibration plate. Move as less as possible.
6. Calibration and running seahorse assay:
  1. Load assay template in Seahorse XF24 software.
  2. Press green ‘START’ button.
  3. Make sure to load the correct protocol, the correct save directory and saving name.
  4. Press ‘START’.
  5. Load sensor cartridge with calibration plate into instrument tray (the notch goes in the front, left corner. Make sure that the plate sits correctly and flat, between all 8 tabs)
  6. Follow the instructions on the screen in order to calibrate and equilibrate sensors.
  7. Once equilibration step is done, remove the calibration plate and replace with cell culture plate.
Protocol commands (mitochondria stress test, Table 2, Figure 5)

Table 2. Protocol commands for mitochondrial stress test

Data analysis

Results were initially reviewed using the seahorse XF data viewer which automatically saves data as MS Excel (.xls) file. Graphic and statistical analyses were carried out using GraphPad Prism. The significance of observed differences of the basal bioenergetics of cell lines was evaluated by the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test. In all cases, P < 0.05 was considered to be significant. Experimental values are reported as mean ± standard deviation (Figure 5) or in a box plot (Figure 6).

Figure 5. Mitochondrial stress test. OCR was measured after mensacarcin was injected (black arrow) in different concentrations to SK-Mel-28 cells followed by consecutive injections of oligomycin (1 μM), FCCP (0.5 μM), and antimycin A (0.5 μM)/rotenone (0.5 μM) (n = 3).

Figure 6. Basal bioenergetic state of SK-Mel-28, SK-Mel-5 and HCT-116 cells. The basal energy metabolism of each cell line was assessed by analyzing OCR/ECAR ratios. OCR and ECAR were acquired with the same protocol as described above but without the injection of compounds. The protocol commands consisted of one loop with 8 measurements. Several separate assays were performed (n = 25).

Notes

Pipet into ports with angle, do not touch the bottom, do not tap to prevent leakage. The liquid is only held by capillary forces.
It is mandatory to load ports for the background wells with assay media that contains the same concentration of DMSO as the compounds to account for any DMSO effects on cells.
Once injected into the wells, compounds are diluted 1:10. This will give a final concentration of 1 µM oligomycin and 0.5 µM FCCP, rotenone and antimycin A, respectively, in the cell culture well.

Recipes

Culture media (10% (v/v) FBS)
Note: Work under sterile conditions in a laminar flow hood.
1. Open liquid DMEM bottle
2. Take out 55 ml with a sterile Serological pipette and discard the liquid
3. Add 50 ml FBS with a sterile Serological pipette
4. Add 5 ml penicillin/streptomycin solution
5. Store at 4 °C
Assay media (sterile, unbuffered, 250 ml)
Note: Work under sterile conditions in a laminar flow hood.
1. Autoclave 250 ml ultrapure H₂O in a glass bottle
2. Dissolve 3,34 g powder DMEM without NaHCO₃ and without HEPES in 250 ml autoclaved H₂O
3. Warm to 37 °C
4. Adjust to pH 7.40 with NaOH (1 M)
5. Store at 4 °C
NaOH (1 M)
Dissolve 4 g NaOH pellets in 100 ml autoclaved H₂O
Oligomycin (10 µM)
1. Prepare freshly on the day of seahorse assay (day 2) (see Note 1)
2. Prepare 1 mM solution in 1 ml DMSO: Dissolve 0.7911 mg oligomycin in DMSO
3. Dilute to 10 µM in assay media (1% DMSO): Pipet 20 µl of 1 mM oligomycin into 1,980 µl assay media
4. Warm to 37 °C and adjust to pH 7.4 with NaOH (1 M) if necessary
FCCP (5 µM)
1. Prepare freshly on the day of seahorse assay (day 2) (see Note 1)
2. Prepare 50 mM solution in DMSO: Dissolve 2.54 mg FCCP in 200 µl DMSO
3. Dilute to 500 µM: Pipet 10 µl of 50 mM FCCP into 990 µl DMSO
4. Dilute to 5 µM in assay media (1% DMSO): Pipet 20 µl of 500 µM FCCP into 1,980 µl assay media
5. Warm to 37 °C and adjust to pH 7.4 with NaOH (1 M) if necessary
Rotenone (5 µM)/antimycin A (5 µM)
1. Prepare freshly on the day of seahorse assay (day 2) (see Note 1)
2. Prepare 50 mM solution in DMSO: Solve 3.94 mg rotenone and 5.49 mg antimycin A in 200 µl DMSO
3. Dilute to 1 mM: Pipet 10 µl of 50 mM rotenone/antimycin A into 490 µl DMSO
4. Dilute to 5 µM in assay media (0.5% DMSO): Pipet 20 µl of 1 mM rotenone/antimycin A into 1,980 µl assay media
5. Warm to 37 °C and adjust to pH 7.4 with NaOH (1 M) if necessary

Acknowledgments

The work was primarily supported by OSU startup funds. B.P. thanks DFG (Deutsche Forschungsgemeinschaft) for postdoctoral funding (PL 799/1-1). We wish to thank Dr. Napur Pande for providing SK-Mel-28 cells, and Bioviotica (Prof. Dr. Axel Zeeck and Hans-Peter Kroll) for providing mensacarcin. We like to thank Elizabeth N. Kaweesa for help with this protocol and Dr. Jeffrey D. Serrill and Prof. Jane E. Ishmael for information and feedback on the seahorse experiments. The authors declare that there are no conflicts of interest or competing interests.

References

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