This article describes a method for isolating immune cells from the bronchoalveolar lavage fluid (BALF) of a mouse model infected with non-typeable Haemophilus influenzae. The procedure involves several steps of fluid collection and cell separation to prepare samples for downstream analysis.
Begin with a euthanized mouse with an exposed trachea. The lungs of the mouse were pre-infected with non-typeable Haemophilus influenzae, a pathogenic bacterium.
Insert a catheter into the trachea and attach it to a syringe filled with wash buffer.
Administer the wash buffer containing an anticoagulant to prevent blood clot formation and a sugar to preserve cell integrity.
Gently aspirate to recover the bronchoalveolar lavage fluid containing red blood cells, immune cells, and residual pathogens.
Repeat the procedure and pool the recovered lavage fluid into a single suspension.
Take an aliquot from the pooled suspension and centrifuge to separate the cells. Discard the supernatant.
Resuspend the cells in a lysis buffer to selectively lyse the red blood cells.
Centrifuge to separate the remaining cells and discard the supernatant containing debris.
Resuspend the remaining cells in a buffer.
The final suspension, containing immune cells and residual bacteria, is ready for downstream analysis.
Expose the ventral part of the lung to access the trachea. Intubate the trachea with a 20 gauge catheter.
Using the catheter, instill 0.8 milliliters of BALF buffer and collect the lavage by dental aspiration. Repeat this procedure three more times, pooling the four lavage collections into a single suspension.
Take a 100 microliter aliquot and spin down for two minutes at 2,000 RPM.
Remove the supernatant and resuspend in RBC lysis buffer. Spin it down and resuspend the pellet with 1X PBS.
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