Can anyone post a protocol for PTT, especially about which pyruvate source to use and its dissolution, concentration. How to calculate the injection volume? One time I did, I had a big issue solubilizing the pyruvate.
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Scale
Beaker for weighing mice
Syringes
2.5g pyruvate in 10 mL PBS- This will correspond to 2.5 g/kg injections.
Timer
Protocol
Remove food from mice for about 6h.
Typically starve the mice at 9AM and aim to start injections at 3 PM
Weigh mice, mark tails if necessary and take fasting glucose measurement via a tail clip.
Prepare pyruvate syringes with 10 uL per g mouse weight (ie for a 30g mouse, 300 uL).
At approximately 1 min intervals, inject appropriate amount of pyruvate into interperitoneal cavity of the mouse.
Immobilize mouse and restrain tail with one hand
Aim needle between the midline and the hip bone
Insert syringe (do not inject) into cavity
Eject syringe.
At desired intervals (normally 15, 30, 45, 60, 75, 90, 105 and 120 min), take blood glucose measurements from tail vein. If needed re-snip the tail vein. When measuring glucose just lift the tail of the mouse, while leaving it in the cage, rather than removing and restraining the mouse.
Analyse data by both % change from fasting glucose and absolute values.
sorry I haven't done those test yet. I hope it is help.
I have never done this test, but I have a suggestion. If you are trying to make a concentrated solution of pyruvate, which is an acid, you should use a salt of it (potassium or sodium) for the sake of solubility, and for pH neutrality.