I was using a metallic probe to have access to the trachea and I inject 500µl of PBS (Phosphate-buffered saline).
Using the Biorad Milliplex Kit to quantify the cytokines in BAL, I have almost nothing in BAL that's why I'm wondering if there was a problem when I perform my BAL.
Do you have any recommendation to optimize my protocol?
Please help! Thank you..
In rats we used two aliquots of 5 ml, PBS and others have used up to 10 aliquots of 5ml.
500microL (0.5ml) may be not enough in mice on a 10:1 proportion. Perhaps you should try up to 5ml in aliquots..
we also made sure the tracheal needle was pushed towards lung.
we got 95% fluid recovery and good alveolar free cell yields .
see attached publn.
Article Silica-Induced Apoptosis in Alveolar and Granulomatous Cells in Vivo
See the article attached "Simvastatin Pretreatment prevents ambient particle-induced lung injury in mice". The standard protocol used in the lab and described in the article stands for 12 lavages of 1 ml each with cold PBS with a metallic probe. However if you are quantifying cytokines in BAL, you only need to dose the first ml lavaged. The result also depends on which cytokine are you studying and the time passed after the stimuli/insult in your experimental design, as every cytokine has an expression profile, sort of a temporal window where they can be detected. Maybe in your experimental design, you have to dose serum instead of BAL to check the systemic levels.
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