TITLE: Mito and Glycolysis Stress Tests for Enteroendocrine Cells - Hutu-80 
AUTHORS: Jan-Willem Taanman, r.mezabrovschi

[DESCRIPTION]
The Seahorse XFp Extracellular Flux Analyzer is designed to compare two different cultures or treatments. Culture cells as usual in either cell culture dishes or flasks as preferred. It is best to compare two cultures of a similar passage number, cell density, time that the cells have been in culture and time since the medium was changed as all this influences mitochondrial respiration. Culture cells until they are almost/just confluent. Cell should have been fed recently before use.

[STEPS]
SECTION: Day 1 - Harvesting, Counting and Diluting
1. Harvest two cell cultures flasks - work in a laminar flow hood.

SECTION: Day 1 - Harvesting, Counting and Diluting
1.1. Aspirate off medium; rinse each flask with 5 mL  of PBS; aspirate off PBS; add 1 mL  of accuitase solution to each flask; incubate 5 min  at 37 °C; add 9 mL of medium to the plate; transfer resuspended cells to a 50-ml centrifuge falcon. 

SECTION: Day 1 - Harvesting, Counting and Diluting
2. Pellet cells by centrifugation 500 x g, 5 min

SECTION: Day 1 - Harvesting, Counting and Diluting
4. When centrifugation of cell suspension is finished, aspirate off supernatants. 
SECTION: Day 1 - Harvesting, Counting and Diluting
3. While centrifuge is spinning, pipet 10 µL of Trypan blue solution in two 0.5-ml tubes placed in a rack.

SECTION: Day 1 - Harvesting, Counting and Diluting
5. Re-suspend cell pellets thoroughly in 5 mL of medium, , by pipetting up and down. 

SECTION: Day 1 - Harvesting, Counting and Diluting
6. Pipet under sterile conditions 10 µL  of the cell suspension into two 0.5-ml tubes with Trypan blue one for each condition. 

SECTION: Day 1 - Harvesting, Counting and Diluting
7. Load 10 µL  of each cell suspension in Trypan blue into the slots of a counting slide. 

SECTION: Day 1 - Harvesting, Counting and Diluting
8. Under a microscope, count the number of cells in 5 of the 9 large squares, average the  counts and write down the cell number. (Each large square has a volume of 0.1 ul).
SECTION: Day 1 - Harvesting, Counting and Diluting
9.  Calculate the number of cells per ul: 

Number of cells per ul = [(number of cells in 5 large squares)/5] x dilution factor x 10
The dilution factor is 2 (1:1 dilution with Trypan blue solution).
Thus: Number of cells per ml (cell density=CD) = number of cells in 5 large squares x 4
SECTION: Day 1 - Harvesting, Counting and Diluting
10. The wells of the XFp Cell Culture Mini-Plate need to be seeded with 20,000 enetoendocrine (EEC) cells in 80 µL  medium. Thus, a cell density of (20,000/80=) 250 cells/ul is required.

Use the equation below to calculate the amount of medium that needs to be added to the cell suspension in the 50-mL centrifuge falcon to obtain the correct density for seeding of the XFp Cell Culture Mini-Plate.
B = [(CD/250) x A] – A
A = the total volume of the cell suspension in the 50 mL  centrifuge falcon (in ml; see step 5)
B = the volume to be added to the 50-mL centrifuge falcon to obtain the correct cell density for seeding of the XFp Cell Culture Mini-Plate (in ml)
CD = the cell density determined with the counting slide (in cells/ml; see step 9)



SECTION: Day 1 - Harvesting, Counting and Diluting
11. To the 50-mL falcon with the cells add the calculated amount of medium to obtain a cell density of 250 cells per ul
SECTION: Day 1 - Harvesting, Counting and Diluting
14. Then in wells A and H of the XFp Cell Culture Miniplates add 80 µL of your cultuing media containing no cells - these are use to calibrate the plate. 

SECTION: Day 1 - Harvesting, Counting and Diluting
15. In wells B-D for condition 1 (usually the control group) and wells E-G for condition 2 add 80 µL of the media now containing the resuspended cell. After seeding the remaining cells can be used to seed new dishes or flasks for further culturing.

SECTION: Day 1 - Harvesting, Counting and Diluting
12. Add 400 µL of the XF Calibrant to each moat of the XFp Cell Culture Miniplate. Once this is done you can place the lid back on and place this in a CO2 incubator   .

SECTION: Day 1 - Harvesting, Counting and Diluting
16. In the XFp Extracellular Flux plate remove the top green section of the cassette and place upside down without touching the sensors (light sensitive) fill each moat with 400 µL of the XF Calibrant and then each well with 200 µL of the XF Calibrant. 

SECTION: Day 1 - Harvesting, Counting and Diluting
13. Re-assemble the  XFp Extracellular Flux plate and place in a humidified 37 °C incubator (no CO)  

SECTION: Day 1 - Harvesting, Counting and Diluting
17. Switch on the Seahorse XFp Extracellular Flux Analyzer with the switch at the back of the machine. It is important to switch on the machine ≥5 h before use. In practice, switch the machine on the day before you use it. The machine is designed to be switched on over long periods of time, so there is no need to switch it off if it is not used for 1‒2 days. 
18. 
SECTION: Day 1 - Harvesting, Counting and Diluting
131. Prepare under sterile conditions in laminar flow hood.
As we routinely perform two mito stress tests on one day, the volumes given below are sufficient for two experiments. If only one experiment is carried out, half the volumes.
0.131. Pipet 20 mL of Seahorse XF DMEM medium, pH 7.4, in a 20-ml tube.

SECTION: Day 2 - Preparation of Assay Medium
19. Pipet 20 mL of Seahorse XF DMEM medium, pH 7.4, in a 20-ml tube.
SECTION: Day 2 - Preparation of Assay Medium
19.1. Add to the tube:         

●   80 µL of 2.5 Molarity (m)d -(+)-glucose
● 200 µLof 100 millimolar (mM)sodium pyruvate
● 200 µLof 200 millimolar (mM) l-glutamine

Mix by vortexing

The XF DMEM medium, pH 7.4, now contains 10 millimolar (mM) d -(+)-glucose, 1 millimolar (mM) sodium pyruvate and 2 millimolar (mM) l-glutamine. This medium is called mito stress test assay medium.

SECTION: Day 2 - Preparation of Assay Medium
20. Warm mito stress test assay medium in a 37 °C water bath. 

Once warmed up, the mito stress test assay medium can be kept at room temperature. The medium can be used for one day. It is not necessary to keep the medium sterile, as it is used within one day.

SECTION: Day 2 - Preparation of Assay Medium
20. Warm glycolysis stress test assay medium in a 37 °C  water bath. 

Once warmed up, the glycolysis stress test assay medium can be kept at room temperature. The medium can be used for one day. It is not necessary to keep the medium sterile, as it is used within one day.

SECTION: Day 2 - Preparation of Assay Medium
19. Pipet 20 mL of Seahorse XF DMEM medium, pH 7.4, in a 20-ml tube.

SECTION: Day 2 - Preparation of Assay Medium
19.1. Add to the tube:         
● 200 µL of 200 millimolar (mM) l-glutamine

Mix by vortexing!

The XF DMEM medium, pH 7.4, now contains 2 millimolar (mM) l-glutamine. This medium is called glycolysis stress test assay medium.

SECTION: Day 2 - Replacing of standard medium with assay medium
21. There is no need to work sterile in the laminar flow hood. It is easier to do this on the lab bench.

Retrieve the XFp cell manipulate(s) Fromm the tissue culture incubator. You may wish to keep the miniplate(s) in the Carrier Tray for ease of handling. 
SECTION: Day 2 - Replacing of standard medium with assay medium
22. Look at the cells under the microscope to:

● Confirm cell health, morphology and purity (no contamination). 
● Ensure that the cells are adhered, and appear as a consistent monolayer. 
● Make sure the background cells (A and H) contain no cells. 
SECTION: Day 2 - Replacing of standard medium with assay medium
23. Wash the cells with assay medium:

Remove all but 20 µLof the culture medium from each well. The small amount of medium is left to keep the cells from drying out. 

SECTION: Day 2 - Replacing of standard medium with assay medium
23.1. Gently add  200 µL of assay medium, then remove the same amount. 

SECTION: Day 2 - Replacing of standard medium with assay medium
23.2. Repeat step 24.1. 
SECTION: Day 2 - Replacing of standard medium with assay medium
23.3. Add 155 µL to a total volume of 175 µL per well. 

SECTION: Day 2 - Replacing of standard medium with assay medium
24. Observe the assay wells under a microscope to ensure that the cells were not washed away. 
SECTION: Day 2 - Replacing of standard medium with assay medium
25. Place the plate in a humidified 37 °C  incubator without CO2 for 60 minprior to the assay. 

SECTION: Day 2 - Replacing of standard medium with assay medium
21. There is no need to work sterile in the laminar flow hood. It is easier to do this on the lab bench.

Retrieve the XFp miniplate(s) from the tissue culture incubator. You may wish to keep the miniplate(s) in the Carrier Tray for ease of handling. 
SECTION: Day 2 - Replacing of standard medium with assay medium
22. Look at the cells under the microscope to:

● Confirm cell health, morphology and purity (no contamination). 
● Ensure that the cells are adhered, and appear as a consistent monolayer. 
● Make sure the background cells (A and H) contain no cells. 
SECTION: Day 2 - Replacing of standard medium with assay medium
23. Wash the cells with assay medium:

Remove all but 20 µLof the culture medium from each well. The small amount of medium is left to keep the cells from drying out. 

SECTION: Day 2 - Replacing of standard medium with assay medium
23.1. Gently add  200 µL of assay medium, then remove the same amount. 

SECTION: Day 2 - Replacing of standard medium with assay medium
23.2. Repeat step 24.1. 
SECTION: Day 2 - Replacing of standard medium with assay medium
23.3. Add 155 µL to a total volume of 175 µL per well. 
SECTION: Day 2 - Replacing of standard medium with assay medium
24. Observe the assay wells under a microscope to ensure that the cells were not washed away. 
SECTION: Day 2 - Replacing of standard medium with assay medium
25. Place the plate in a humidified 37 °C incubator without CO2 for 60 minprior to the assay. 

SECTION: Day 2 - Preparation oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 solutions for a mito stress test
26. Take the 10 millimolar (mM) oligomycin A, 10 millimolar (mM) FCCP, 10 millimolar (mM) rotenone and 10 mM antimycin stock solutions out of the -20 °C freezer and put on ice.  Also take the 20 millimolar (mM)  Hoechst 33342  stock solution out of the fridge and put on ice.

SECTION: Day 2 - Preparation oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 solutions for a mito stress test
27. Pipette 1 mL of mito stress test assay medium each in three 1.5-ml tubes and place in a rack.

SECTION: Day 2 - Preparation oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 solutions for a mito stress test
28. Label the tubes with “Oligo”, “FCCP” and “Rot + AA”.
SECTION: Day 2 - Preparation oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 solutions for a mito stress test
29. Pipette 5 µL of 10 millimolar (mM) oligomycin A into the tube labelled “Oligo” to make a 50 micromolar (µM) solution.

SECTION: Day 2 - Preparation oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 solutions for a mito stress test
30. Pipette 5 µL of 10 millimolar (mM) FCCP into the tube labelled “FCCP” to make a 50 micromolar (µM)solution.

SECTION: Day 2 - Preparation oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 solutions for a mito stress test
31. Pipette 5 µLof 10 millimolar (mM) rotenone and 5 µLof 10 millimolar (mM) antimycin A into the tube labelled “Rot + AA” to make a 50 micromolar (µM) + 50 micromolar (µM)solution.

SECTION: Day 2 - Preparation oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 solutions for a mito stress test
32. Vortex the three tubes briefly.
SECTION: Day 2 - Preparation oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 solutions for a mito stress test
33. Label three new 1.5-ml tubes with “Oligo 2”, “FCCP 2” and “Rot + AA + H”.
SECTION: Day 2 - Preparation oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 solutions for a mito stress test
34. Pipette 408 µL of mito stress test assay medium in the tube labelled “oligo 2” and add 192 µLof 50 micromolar (µM) oligomycin A.

SECTION: Day 2 - Preparation oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 solutions for a mito stress test
35. Pipette 438 µLof mito stress test assay medium in the tube labelled “FCCP 2” and add 162 µL of 50 micromolar (µM) FCCP.

SECTION: Day 2 - Preparation oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 solutions for a mito stress test
36. Pipette 380 µL of mito stress test assay medium in the tube labelled “Rot + AA +H” and add 100 µL of 50 micromolar (µM) rotenone + 50 micromolar (µM) antimycin A. Then add 20 µL of 20 millimolar (mM)Hoechst 33342 stock solution. 

SECTION: Day 2 - Preparation oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 solutions for a mito stress test
37. Vortex the three tubes briefly.
 
NB: The final concentrations in the wells of the XFp cell miniplate are: 2 micromolar (µM) oligomycin A, 1.5 micromolar (µM) FCCP,1 micromolar (µM) rotenone,1 micromolar (µM)antimycin A and 66 micromolar (µM) Hoechst 33342.

SECTION: Day 2 - Preparation glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 solutions for a glycolysis stress test
26. Take the 10 millimolar (mM) oligomycin A and 500 millimolar (mM) 2-deoxyglucose stock solutions out of the -20 °C freezer and put on ice. Also take the 20 millimolar (mM) Hoechst 33342 stock solution out of the fridge and put on ice. Finally, take the 2.5 Molarity (M) glucose solution from the shelf and put in a rack.

SECTION: Day 2 - Preparation glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 solutions for a glycolysis stress test
27. Pipette 1 ml of glycolysis stress test assay medium each in two 1.5-ml tubes and place in a rack.
SECTION: Day 2 - Preparation glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 solutions for a glycolysis stress test
28. Label the tubes with “GLU” and “Oligo”,and label a third tube with “2-DG + H”.
SECTION: Day 2 - Preparation glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 solutions for a glycolysis stress test
29. Pipette 5 µLof 10 millimolar (mM) oligomycin A into the tube labelled “Oligo” to make a 50 millimolar (mM) solution.

SECTION: Day 2 - Preparation glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 solutions for a glycolysis stress test
30. Vortex the “Oligo” tube briefly.
SECTION: Day 2 - Preparation glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 solutions for a glycolysis stress test
31. Label a new 1.5-ml tube with “Oligo 2”.
SECTION: Day 2 - Preparation glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 solutions for a glycolysis stress test
32. Pipette 336 µL of glycolysis stress test assay medium in the tube labelled “Oligo 2” and add 264 µL of 50 millimolar (mM) oligomycin A.

SECTION: Day 2 - Preparation glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 solutions for a glycolysis stress test
33. Pipette sterile, in the laminar flow hood, 32 µL of 2.5 Molarity (M) glucose into the tube labelled “GLU” with 1 mL of glycolysis stress medium to make an 80 millimolar (mM) glucose solution.

SECTION: Day 2 - Preparation glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 solutions for a glycolysis stress test
34. Pipette 242 µL of the500 millimolar (mM) 2-deoxyglucose stock in the empty tube labelled “2DG + Hoechst” and add 8 µLof20 millimolar (mM) Hoechst 33342 stock solution.

SECTION: Day 2 - Preparation glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 solutions for a glycolysis stress test
35. Vortex the three tubes briefly.
 
NB: The final concentrations in the wells of the XFp cell miniplate are: 10 millimolar (mM) glucose, 2 micromolar (µM)oligomycin A, 50 millimolar (mM) 2-deoxyglucose and 64 micromolar (µM)Hoechst 33342.

SECTION: Day 2 - Preparation oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 solutions for a mito stress test
38. Eliminate air bubbles from the XFp Extracellular Flux Cartridge/Plate. 
SECTION: Day 2 - Preparation oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 solutions for a mito stress test
38.1. Following the overnight incubation, remove the cartridge assembly from the incubator. 
SECTION: Day 2 - Preparation oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 solutions for a mito stress test
38.2. Lift the sensor cartridge completely out of the calibrant and utility plate. 
SECTION: Day 2 - Preparation oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 solutions for a mito stress test
38.3. Immediately return the sensor cartridge back onto the utility plate, submerging the sensors in calibrant. This step helps eliminate any bubbles that may form during overnight hydration. 
SECTION: Day 2 - Preparation glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 solutions for a glycolysis stress test
36. Eliminate air bubbles from the XFp Extracellular Flux Cartridge/Plate. 
SECTION: Day 2 - Preparation glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 solutions for a glycolysis stress test
36.1. Following the overnight incubation, remove the cartridge assembly from the incubator. 
SECTION: Day 2 - Preparation glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 solutions for a glycolysis stress test
36.2. Lift the sensor cartridge completely out of the calibrant and utility plate. 
SECTION: Day 2 - Preparation glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 solutions for a glycolysis stress test
36.3. Immediately return the sensor cartridge back onto the utility plate, submerging the sensors in calibrant. This step helps eliminate any bubbles that may form during overnight hydration. 
SECTION: Day 2 - Loading of the XFp Sensor Cartridge with oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 for the mito stress test
39. Orient the XFp Assay Cartridge: Place well labels (letters A-H) to the left. The triangular notch will be in the bottom left-hand corner. 
SECTION: Day 2 - Loading of the XFp Sensor Cartridge with oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 for the mito stress test
40. Using a p200μL pipette, make sure the tip is securely fitted onto the pipette. Pipette 25 µL of the oligomycin A solution from the tube labelled “oligo 2” in all eight ports A.  

SECTION: Day 2 - Loading of the XFp Sensor Cartridge with oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 for the mito stress test
41. Pipette 25 µLof the FCCP solution from the tube labelled “FCCP 2” in all eight ports B.  

SECTION: Day 2 - Loading of the XFp Sensor Cartridge with oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 for the mito stress test
42. Pipette 25 µL of the rotenone + antimycin A + Hoechst 33342 solution from the tube labelled “rot + AA + H” in all eight ports C. 

Port D remains empty.
SECTION: Day 2 - Loading of the XFp Sensor Cartridge with glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 for the glycolysis stress test
37. Orient the XFp Assay Cartridge: Place well labels (letters A-H) to the left. The triangular notch will be in the bottom left-hand corner.
SECTION: Day 2 - Loading of the XFp Sensor Cartridge with glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 for the glycolysis stress test
38. Using a p200 μL pipette, make sure the tip is securely fitted onto the pipette. Pipette 25 µL of the glucose solution from the tube labelled “GLU” in all eight ports A.  

SECTION: Day 2 - Loading of the XFp Sensor Cartridge with glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 for the glycolysis stress test
39. Pipette 25 µLof the oligomycin A solution from the tube labelled “Oligo 2” in all eight ports B.  

SECTION: Day 2 - Loading of the XFp Sensor Cartridge with glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 for the glycolysis stress test
40. Pipette 25 µLof the 2-deoxyglucose + Hoechst 33342 solution from the tube labelled “2DG + H” in all eight ports C. 

Port D remains empty.
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
43. Touch the screen and Press “Start” on the screen of the XFp Extracellular Flux Analyzer.
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
44. Press “Cell Mito Stress Test”
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
45. Press “>” (bottom right of screen).
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
46. Repeat step 46. 
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
47. Press “Start Assay”. After some time, the plate holder will come out of the machine.
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
48. Carefully transfer the cartridge/utility plate assembly to the plate holder of the XFp Analyzer according to the orientation as indicated on the screen.

IMPORTANT: Remove the plate lid before inserting the cartridge into the machine.
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
49. Press “Continue”. 

The calibration of the XFp Assay Cartridge will take ~20 min. After the calibration is completed, the screen will indicate that the machine is ready to receive the XFp Cell Culture Miniplate.

SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
50. Press “Continue”.     

Take the XFp Cell Culture Miniplate from the 37 °C incubator and replace the utility plate with the XFp Cell Culture Miniplate as indicated on the screen. 

IMPORTANT: Remove the plate lid before inserting the cartridge into the machine.
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
51. Press “Continue”.

Throw away utility plate. The equilibration and measurements will take ~80 min. 

During this time, you can replace the standard medium of a next plate with mito stress test assay medium and incubate the next plate for ≥1 h in the humidified 37 °C  incubator (no CO2).
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
52. About 5–10 min before the assay is finished, you can eliminate air bubbles from the next XFp Sensor Cartridge and load the Cartridge with oligomycin A, FCCP and rotenone + antimycin A + Hoechst 33342 for the mito stress.
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
53. After the measurements have been taken, follow the instructions on the screen. This will ensure that the data are automatically stored on the memory stick under a distinctive name consisting of the date, a unique number and the type of test.
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
41. Touch the screen and Press “Start” on the screen of the XFp Extracellular Flux Analyzer.
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
42. Press “Glycolysis stress test”. 
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
43. Press “>” (bottom right of screen).
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
44. Repeat step 44. 
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
45. Press “Start Assay”. After some time, the plate holder will come out of the machine.
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
46. Carefully transfer the cartridge/utility plate assembly to the plate holder of the XFp Analyzer according to the orientation as indicated on the screen.

IMPORTANT: Remove the plate lid before inserting the cartridge into the machine.
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
47. Press “Continue”. 

The calibration of the XFp Assay Cartridge will take ~20 min. After the calibration is completed, the screen will indicate that the machine is ready to receive the XFp Cell Culture Miniplate.

SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
48. Press “Continue”.    

Take the XFp Cell Culture Miniplate from the 37 °C incubator and replace the utility plate with the XFp Cell Culture Miniplate as indicated on the screen. 

IMPORTANT: Remove the plate lid before inserting the cartridge into the machine.
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
49. Press “Continue”.

Throw away utility plate. The equilibration and measurements will take ~80 min. 

During this time, you can replace the standard medium of a next plate with glycolysis stress test assay medium and incubate the next plate for ≥1 h in the humidified 37 °C incubator (no CO2).
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
50. About 5–10 min before the assay is finished, you can eliminate air bubbles from the next XFp Sensor Cartridge and load the Cartridge with glucose, oligomycin A, and 2-deoxyglucose + Hoechst 33342 for the glycolysis stress test.
 
SECTION: Day 2 - Calibration and measurements with the XFp Extracellular Flux Analyzer
51. After the measurements have been taken, follow the instructions on the screen. This will ensure that the data are automatically stored on the memory stick under a distinctive name consisting of the date, a unique number and the type of test.
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
54. Switch on the BioTek Cytation-1 Cell Imaging Multimode Reader and computer - may require booking out in advance. Let the reader and computer start up (~5 min).

SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
55. Take the Miniplate out of the Seahorse machine.
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
56. Place the Miniplate in the metal adaptor (very snug fit) and place in the plate holder of the BioTek Cytation-1 Cell Imaging Multimode Reader with well A at the top (see picture).

  

SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
57. Start the Gen5 programme by clicking on the icon on the screen.
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
58. Open the existing protocol “JWT 8 well seahorse DAPI only” in the Task Manager window. (Cancel saving “Experiment1”.)
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
59. Click “OK” when the plate is loaded in the plate holder to start the analysis. There should not be a lid on the miniplate but, on the screen, the box “Use lid” can be checked and let the machine record the images (~5 min).

SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
60. Save the Excel file to save the images.
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
61. Click on an image of a well to open the analyses window - in the Gen5 software.
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
62. Deselect the “SEAHORSE: bright Field” box, so that only the “SEAHORSE DAPI” box is selected. Then Click on “Analyse” (middle top of the screen).
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
63. Select “I want to edit an existing Image Analysis step”.
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
64. Click on “Cellular Analysis Cell Count” and click “OK”. Then click on “Options”.
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
65. Make changes so that most nuclei are counted. Here we checked the “Auto” box (top left of screen). Changed the min object size to 5um and the max object size to 40um, in the advanced setting we also changed the image smoothing strength to 1. Then click on “APPLY CHANGES”.
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
66. The cell count is given on the top right side of the screen. Close window (“X”, top right of the screen). Write down cell numbers for each well. Multiply the cell count by 10 to get the number of cells per well. This is used to normalise the data in the Wave Programme. 
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
52. Switch on the BioTek Cytation-1 Cell Imaging Multimode Reader and computer - may require booking out in advance. Let the reader and computer start up (~5 min ).

SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
53. Take the Miniplate out of the Seahorse machine.
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
54. Place the Miniplate in the metal adaptor (very snug fit) and place in the plate holder of the BioTek Cytation-1 Cell Imaging Multimode Reader with well A at the top (see picture).
  

SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
55. Start the Gen5 programme by clicking on the icon on the screen.
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
56. Open the existing protocol “JWT 8 well seahorse DAPI only” in the Task Manager window. (Cancel saving “Experiment1”.)
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
57. Click “OK” when the plate is loaded in the plate holder to start the analysis. There should not be a lid on the miniplate but, on the screen, the box “Use lid” can be checked and let the machine record the images (~5 min ).

SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
58. Save the Excel file to save the images.
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
59. Click on an image of a well to open the analyses window - in the Gen5 software.
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
60. Deselect the “SEAHORSE: bright Field” box, so that only the “SEAHORSE DAPI” box is selected. Then Click on “Analyse” (middle top of the screen).
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
61. Select “I want to edit an existing Image Analysis step”.
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
62. Click on “Cellular Analysis Cell Count” and click “OK”. Then click on “Options”.
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
63. Make changes so that most nuclei are counted. Here we checked the “Auto” box (top left of screen). Changed the min object size to 5um and the max object size to 40um, in the advanced setting we also changed the image smoothing strength to 1. Then click on “APPLY CHANGES”.
SECTION: Day 2 - Determination of cell number with the BioTek Cytation-1 Cell Imaging Multimode Reader after Seahorse experiment
64. The cell count is given on the top right side of the screen. Close window (“X”, top right of the screen). Write down cell numbers for each well. Multiply the cell count by 10 to get the number of cells per well. This is used to normalise the data in the Wave Programme.