macrophage purification from broncho alveolar lavage

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Akemi
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macrophage purification from broncho alveolar lavage

Hi,

Does anyone has experience in isolating macrophage from brochoalveolar lavage? I am interested in the bead separation,

thanks,
a

TeraM
TeraM's picture
Hello,

Hello,
For the isolation of macrophages from BAL you could use a direct selection method using CD14 MicroBeads. For a possible higher purity you could also try a negative selection system.
For isolation of untouched alveolar macrophages from this cell preparation, you could consider labeling the cells with CD3, CD15 and CD236 for depletion…this will all depend on the composition of the BAL.

In all cases a good single cell suspension is important. To achieve this perhaps the following could work for you:
Gravity filter the BAL thru a cell strainer (100um nylon filter—Falcon 352360) into a 50ml conical tube. If the sample is particularly sticky, the filter must be tilted on the top of the tube so that adequate filtration happens. After filtration, centrifuge BAL 10-15 minutes at 300xg. Save the supernatant and wash the cells one time in buffer (PBS/2mM EDTA/0.5% BSA) for 10min at 300xg. Aspirate off all the supernatant and loosen cell pellet. Resuspend cell in small volume to determine count and viability and then continue with labeling protocol.

Regards,
Tera