Ketone Body Tolerance Test: Difference between revisions

Line 9:

*1.5 mg/mL bOHB in PBS (make as 15g bOHB in 10mL of PBS, sterile filtered). This will correspond to 1 U/kg injections. If you are using a higher or lower dose of bOHB, add more or less to the 10 mL of PBS, so that injections are 10 uL/g of mass.

*This may need to be adjusted depending on the ketone sensitivity of the mice, and this is based on PGC-1a on chow.

*In general you want the bOHB to increase blood bOHB levels by about 10 to 12-fold (bringing bOHB levels form around .5mmol/L up to the 5 to 6 mmol/L range in the most responsive of your two groups. If your response is lower or higher, you will probably have to change your dose and retry.

*In general you want the bOHB to increase blood bOHB levels by about 10 to 12-fold (bringing bOHB levels form around .5mmol/L up to the 5 to 6 mmol/L range if fasted or 1.5 to 2.5 mmol/L if fed in the most responsive of your two groups. If your response is lower or higher, you will probably have to change your dose and retry.

*Timer

'''Protocol'''

*If testing fasted ketone tolerance: Remove food from mice for about 6h by putting them in a fresh cage. Add “do not feed” acetate to cages, and ideally move cage to procedure room. Try to make sure that the mice are in a quiet, undisturbed temperature controlled room with the lights on.

*'''If''' testing fasted ketone tolerance: Remove food from mice for about 6h by putting them in a fresh cage. Add “do not feed” acetate to cages, and ideally move cage to procedure room. Try to make sure that the mice are in a quiet, undisturbed temperature controlled room with the lights on. Typically starve the mice at 8AM and aim to start injections at 2PM

**Typically starve the mice at 8AM and aim to start injections at 2PM

*Prepare a 1 g/10mL solution of glucose in the unlikely case that some animals become hyperketonemic.

*Weigh/MRI mice ahead of time, mark tails if necessary with different colors for rapid identification and take fasting ketone measurement via a tail clip.

*Prepare bOHB syringes with 1.5 g/KG mouse weight (ie for a mouse with 15g lean body mass, use 15uL).

*At approximately 1 min intervals, inject appropriate amount of bOHB into intraperitoneal cavity of the mouse.

*Pre-open enough ketone test strips for one round of measurements.

*At approximately 40 sec to 1 min intervals, inject appropriate amount of bOHB into intraperitoneal cavity of the mouse.

**Immobilize mouse and restrain tail with one hand.

**Aim needle for peritoneal space, between the midline and the hip bone.

@@ Line 9: / Line 9: @@
 *1.5 mg/mL bOHB in PBS (make as 15g bOHB in 10mL of PBS, sterile filtered). This will correspond to 1 U/kg injections. If you are using a higher or lower dose of bOHB, add more or less to the 10 mL of PBS, so that injections are 10 uL/g of mass.
 *This may need to be adjusted depending on the ketone sensitivity of the mice, and this is based on PGC-1a on chow.
-*In general you want the bOHB to increase blood bOHB levels by about 10 to 12-fold (bringing bOHB levels form around .5mmol/L up to the 5 to 6 mmol/L range in the most responsive of your two groups. If your response is lower or higher,  you will probably have to change your dose and retry.
+*In general you want the bOHB to increase blood bOHB levels by about 10 to 12-fold (bringing bOHB levels form around .5mmol/L up to the 5 to 6 mmol/L range if fasted or 1.5 to 2.5 mmol/L if fed in the most responsive of your two groups. If your response is lower or higher,  you will probably have to change your dose and retry.
 *Timer
 '''Protocol'''
-*If testing fasted ketone tolerance: Remove food from mice for about 6h by putting them in a fresh cage. Add “do not feed” acetate to cages, and ideally move cage to procedure room. Try to make sure that the mice are in a quiet, undisturbed temperature controlled room with the lights on.
+*'''If''' testing fasted ketone tolerance: Remove food from mice for about 6h by putting them in a fresh cage. Add “do not feed” acetate to cages, and ideally move cage to procedure room. Try to make sure that the mice are in a quiet, undisturbed temperature controlled room with the lights on. Typically starve the mice at 8AM and aim to start injections at 2PM
-**Typically starve the mice at 8AM and aim to start injections at 2PM
 *Prepare a 1 g/10mL solution of glucose in the unlikely case that some animals become hyperketonemic.
 *Weigh/MRI mice ahead of time, mark tails if necessary with different colors for rapid identification and take fasting ketone measurement via a tail clip.
 *Prepare bOHB syringes with 1.5 g/KG mouse weight (ie for a mouse with 15g lean body mass, use 15uL).
-*At approximately 1 min intervals, inject appropriate amount of bOHB into intraperitoneal cavity of the mouse.
+*Pre-open enough ketone test strips for one round of measurements.
+*At approximately 40 sec to 1 min intervals, inject appropriate amount of bOHB into intraperitoneal cavity of the mouse.
 **Immobilize mouse and restrain tail with one hand.
 **Aim needle for peritoneal space, between the midline and the hip bone.