Extracellular Flux Assay in 3D Spheroids: A Method for Measuring the Metabolic Activity of Pancreatic Tumor Spheroids

Overview

This protocol describes the technique for performing extracellular flux assay on 3D spheroids generated from the co-culture of pancreatic cancer cells (Patu8902) and activated pancreatic fibroblasts (PS1) to measure the two major energy-producing pathways, glycolysis and mitochondrial respiration.

Protocol

1. Metabolic Assay of 3D Pancreatic Tumor Spheroids for Bioenergetics Pathways using the Extracellular Flux Analyzer

NOTE: In this section, the analysis of the metabolic functions of the spheroids using an extracellular flux analyzer with assay microplates specifically designed for 3D spheroids is described.

1. Washing and Transfer of spheroids from growth plates to spheroid assay microplates.
   NOTE: Spheroids may be used for metabolic assays when they reach a diameter of ~500 µm.
   1. The day prior to performing the assay, hydrate the probe or sensor cartridge with assay calibrant (200 µL/well) in the provided utility plate and incubate overnight (4-18 h) in a humidified incubator (non-CO₂) set at 37 °C.
   2. Prepare the appropriate assay medium for the desired metabolic assay by supplementing the base media (see the Table of Materials) with either 2 mM L-glutamine for glycolysis stress test (GST) assay or with 1 mM pyruvate, 2 mM L-glutamine and 10 mM glucose for a mitochondrial stress test (MST) assay.
      NOTE: The extracellular flux analyzer measures both mitochondrial respiration (OCR) and glycolysis (ECAR) of live cells in a 96-well plate format. These rates provide an overview of cellular metabolic function in samples under investigation. Please refer to the manual in the assay kits for detailed instructions.
   3. After adding assay specific supplements (refer Step 1.1.1), warm the supplemented assay medium in a 37°C water bath and adjust the pH to 7.4 ± 0.1 with 0.1N NaOH. Keep the medium warm until use.
   4. Reconstitute assay kit reagents in the calibrated assay medium to recommended stock concentrations.
      NOTE: These are made at 10× the final concentration desired in the wells. Stock concentrations for glucose, oligomycin and 2-deoxy-glucose (2-DG) are 100 mM, 100 µM and 500 mM, respectively.
   5. Observe spheroids in the growth plate under a light microscope to check for spheroid morphology and overall uniformity; in terms of the shape and structure of the spheroids.
   6. Transfer the growth plate onto a magnetic holding drive and carefully aspirate ~120 µL growth media. Gently wash spheroids with ~120 µL of warmed and pre-calibrated assay media, aspirate and repeat the wash twice. Inspect the spheroids under the microscope again to ensure that spheroids are not washed away.
   7. Prepare the spheroid assay microplate by adding 180 µL warm assay media to each well.
   8. Set a P20 micropipette to 10 µL and fit a wide bored tip to it. If wide bored tips are not available, cut off the tips of regular pipette tips with a clean scissors or scalpel to widen the bore.
      NOTE: This should reduce shearing and preserve the overall morphology of the spheroids while being transferred to assay plates.
   9. Use a warm (37°C) surface to carry out transfer of spheroids. Use an X-ray film viewer surface warmed with a white-light lamp to enhance contrast and aid visualization of spheroids during the transfer process.
   10. Carefully aspirate a single pre-washed spheroid from the cell repellent growth plate using a P20 micropipette fitted with a wide bore tip and gently transfer spheroid directly into the center of each well of a spheroid assay plate for carrying out the metabolic assay. Allow each spheroid to gently fall into the central micro-chamber of each well by pure gravity to populate the assay plate.
       NOTE: It usually takes 5-8 s for each spheroid to fall into the center of the well by gravity. Do not pull out the pipette until the spheroids have settled down in the micro-chamber. Do not deposit spheroids in the 4 corner wells of the assay plate (A1, A12, H1, H12) since these will be used as background wells.
   11. Monitor the morphology and position of each spheroid to ensure that each spheroid is in the center of each well. If necessary, reposition to the center of well by aspirating out spheroid using a wide bore pipet tip, and subsequent deposition by gravity.
   12. Once all spheroids have been transferred, place the assay plate in a 37 °C humidified air incubator (non-CO₂) for one hour prior to assay.
2. Loading sensor cartridge with compounds and performing the assay
   1. Prepare 10× concentrated assay specific reagents in the assay specific media described in step 1.1.2. For example, to carry out GST, reconstitute glucose, oligomycin, and 2-DG in recommended volumes referred to in the assay manual. Both GST and MST assays were carried out using Patu8902-PS1 spheroids.
   2. Properly orient the sensor cartridge with rows labeled A-H on the left hand side and place a loading guide on top of the sensor cartridge. Ensure correct loading of desired port by orienting the loading guide so that the letter corresponding to the port to be loaded is at the upper left hand corner.
   3. Using a multichannel pipette, dispense reagents directly into injection ports. Hold loading guides in position using fingertips throughout the procedure.
      NOTE: Each reagent will be injected in a recommended port referred in the assay manual. Avoid creating air bubbles but do not tap any part of the cartridge to remove air bubbles.
   4. Remove loading guide and position at eye level with cartridge to visually inspect injection ports for even loading.
   5. Create the experimental assay design/instrument protocol using the instrument controller Wave software (henceforth, analysis software) as described in section 1.3.
3. Assay design and execution using the analysis software
   1. Create an assay template for the experiment
      1. Open the analysis software and click "Templates" or choose from an existing assay template. Double click on "Blank Template".
      2. Define experimental groups and conditions.
         1. Define Injection strategies for ports A, B, and C. Leave port D empty.
            NOTE: For GST assay, only these ports will be loaded with Glucose, Oligomycin and 2-DG. In case of MST assay, oligomycin, FCCP and rotenone will be loaded.
         2. Define pre-treatments if spheroids were treated with one or more test compounds and the control group. Define the assay media to include source, supplements, and other information.
         3. Define one or more cell type (e.g. Patu8902, PS1) seeing the density and passage number information.
      3. Click "Generate Groups".
      4. Click "Plate Map" tab and assign groups to the assay plate corresponding to the experimental design.
      5. Select the four corner wells as background wells. Ensure that there are no spheroids in these wells.
4. Run assay on the analyzer
   1. Click on the Instrument Protocol tab. Keep the default protocol commands by checking "Calibrate", "Equilibrate" and "Baseline Measurement Cycle".
   2. Click "Injections" and define compound for each port. Edit measurement cycles to 6 cycles from the default 3 cycles for spheroids. Keep the default 3 min mix, 0 min wait, and 3 min measure times.
      NOTE: Default mix-wait-measure times are 3 min, 0 min, 3 min, respectively. Three basal rate measurements are typically taken prior to injection of first assay compound. These measurements can be calibrated and readjusted as per the experimental design.
   3. Review Protocol and Group Summary.
   4. Save assay design template.
   5. Proceed to pre-assay calibration once injection ports have been loaded with assay substrates. Transfer the cartridge with the utility plate to the extracellular flux analyzer.
      NOTE: This usually completes within 15-20 min and calibrates the sensors to conduct OCR and ECAR measurements.
   6. After calibration of the cartridge, replace the utility plate with the pre-warmed assay plate containing 3D spheroids and initiate the assay. After the measurements are completed, export the data to spreadsheet or graphing and statistical software to analyze the data.

Materials

Name	Company	Catalog Number	Comments
96 well cell repellant plate	USA Scientific/ Griener Bio-One	655970	spheroid growth plate
D-Glucose	Sigma	D9434
DPBS	Gibco	14190-144
Glycolysis Stress Test Kit	Agilent Technologies	103020-100
L-Glutamine	Sigma	59202C
Mitochondrial Stress Test Kit	Agilent Technologies	103015-100
Patu8902 cells	Laboratory Stock		pancreatic ductal adenocarcinoma cells
PS1 cells	Laboratory Stock		activated pancreatic stellate cells
RPMI1640	Gibco	11875-093	growth media for cells, spheroids
SeaHorse XFe 96 extracellular flux analyzer	Agilent Technologies		extracellular flux analyzer
Sodium Pyruvate	Sigma	S8636
XF Base media	Agilent Technologies	102365-100	base media for metabolic assays on XFe 96