TITLE: Seahorse protocol for islets using Xfe24 Analyzer
AUTHORS: Nancy Smith

[DESCRIPTION]
Using Agilent's seahorse Xfe Analyzer with the islet capture microplates to assess whole islet bioenergentics in vitro.  Agilent Seahorse XFe24 Analyzers measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells in a 24-well plate format.

[STEPS]
SECTION: Solution Prep - Calibrant Solution
1. Calibrant Solution (Agilent 100840-000, 500ml) is provided by Agilent and stored at room temperature.


SECTION: Solution Prep - MA media
2. Use the DMEM (Agilent 103575-100, 500ml) provided by Agilent; stored at 4 oC. Needs to be supplement with 1% Fetal Bovine Serum (FBS, Gibco 12483-020).  Add 5ml of heat inactivated FBS to the 500ml DMEM.  Filter sterilize and store at 4oC.


SECTION: Solution Prep - Stocks
3. Glucose 2M stock in Dimethyl sulfoxide (DMSO, Fisher D128-1) - A
Weigh 18.2g of glucose into 50ml DMSO.  Aliquot and store at -20oC.
SECTION: Solution Prep - Stocks
4. Oligomycin (Sigma O4876-5MG)  5mM stock in DMSO - B
Pipette 1.27ml DMSO to the 5mg Oligomycin.  Aliquot and store at -20oC.
SECTION: Solution Prep - Stocks
5. FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, Sigma C2920-10MG) 1mM stock in DMSO - C
Prepare 50mM stock of FCCP by adding 787ul of DMSO to the vial containing 10mg of FCCP.  To make 1mM stock, use 20ul of the 50mM FCCP into 980ul of DMSO. Aliquot the 1mM stock and store both stocks at -20oC.
SECTION: Solution Prep - Stocks
6. Rotenone (Sigma R8875-5G)  5mM stock in DMSO - D
Weigh 1.97g of Rotenone into 1ml of DMSO.  Aliquot and store at -20oC.
SECTION: Solution Prep - working stocks
8. Glucose (167mM) - A
Pipette 167µl of 2M glucose stock to 1833µl MA media.  Working stock concentration of 167mM with an injected final concentration of 16.7mM. 


SECTION: Solution Prep - working stocks
9. Oligomycin (55uM) - B
Pipette 22µl of 5mM oligomycin stock 
to 1978 µl MA media.  Working stock concentration of 55uM with an injected final concentration of 5µM. 
 
SECTION: Solution Prep - working stocks
10. FCCP - C
Mouse (55.5µM)
Pipette 111 µl of 1mM FCCP stock to 1889 µl MA media.  Working stock concentration of 55.5µM with an injected final concentration of 5µM. 
Human (33.3µM)
Pipette 66.6 µl of 1mM FCCP stock to 1933.4 µl MA media.  Working stock concentration of 33.3µM with an injected final concentration of 3µM. 
     **FCCP needs to be titrated for every lot**
SECTION: Solution Prep - working stocks
11. Rotenone (55.2 µM)/Antimycin A (56.2 µM) - D
Pipette 22.5 µl of 5mM rotenone stock, 22.5 µl of 5mM antimycin A stock to 1955 µl MA media.  Working stock concentration of 55.2 µM with an injected final concentration of 5µM 
SECTION: Solution Prep - Stocks
7. Antimycin A (Sigma A8674-25MG) 5mM stock in DMSO - D
Weigh 2.7mg of Antimycin A into 1ml DMSO. Aliquot and store -20oC.
SECTION: Day one Protocol
12. The day before the assay, add 1 ml of calibrant solution to each of the wells of the utility plate of the extracellular flux
assay kit. 
 
 
Incubate the entire set of plates at 37oC with NO CO2 overnight.

 

SECTION: Day two Protocol
13. Warm MA media in waterbath to 37oC.

SECTION: Day two Protocol
15. Place islets (min 70 islets per well), into tube with 500 µl of MA media.  Let islets gravity settle to the bottom of the tube.  Pipette islets (100µl ) and carefully dispense into center of the well of islet capture microplate.  Place screen, flat side down, to well using forceps ensuring that the screens click into the well.  Ensure  there are no bubbles trapped under the screens.  Add the remaining MA media to a total volume of 500 µl in each well (by pipetting onto the side of the well).  Incubate at 37oC with NO CO2 for 1hr. 
 
 

SECTION: Day two Protocol
14. Place screens into a dish with MA media.
SECTION: Day two Protocol
16. Ensure to not place islets on the wells that are designated as blanks.  See template below.
 

SECTION: Day two Protocol
17. While islets are incubating, turn on seahorse Xfe Analyzer, turn on computer and open Wave.  Open protocol and set up plate details.
Protocol parameters:

 

SECTION: Day two Protocol
19. Load solutions into the ports of the 
extracellular flux assay kit plate.
A - Glucose (50µl)
B- Oligomycin (55µl)
C- FCCP (60µl)
D- Rotenone/Antimycin A (65µl)

 

SECTION: Day two Protocol
20. Press run on the seahorse program.  Place extracellular flux assay plate (with booster plate and lid removed).  Once initialization and 1hr incubation is complete remove the utility plate and replace with islet plate. 
SECTION: Day two Protocol
21. Once complete, remove plate, and collect for DNA or protein.
SECTION: Day two Protocol
18. Make working stocks solutions.
SECTION: Day two Protocol
21.1. Collect islets from well, including scraping off islets from the capture screens into a 1.5ml tube.  Use extra MA media to wash wells
SECTION: Day two Protocol
21.2. Centrifuge tubes at 1000rpm for 4 minutes for protein or 1500rpm for 6 minutes for DNA.
SECTION: Day two Protocol
21.3. Aspirate supernatant.  Store dry pellet at -20oC until ready to run protein or DNA assay.  
SECTION: Solution Prep - MA media
2.1. On the day of experiment, supplement with 2mM or 2.8mM D-glucose (Sigma G8270), 2mM sodium pyruvate (Gibco 11360-070) and 2mM L-glutamine (Gibco 25030-081).  Add 0.018g or 0.025g of glucose to 50ml MA media. Pipette 1ml of (100mM) sodium pyruvate and 500ul of (200mM) L-glutamine to the 50ml MA media.  Mix until glucose has dissolved.

SECTION: Solution Prep - working stocks
8.1. Glucose (200mM) - A
Pipette 200µl of 2M glucose stock to 1800µl MA media.  Working stock concentration of 200mM with an injected final concentration of 20mM. **   
SECTION: Solution Prep - working stocks
10.1. FCCP cat#C2920-10MG, Sigma, lot#0000120405
optimal concentration on mouse islets = 5 µM
optimal concentration on human islets - 3 µM