Identification and Enumeration of Immune Cells in Bronchoalveolar Lavage Fluid by Flow Cytometry

After the third bronchoalveolar lavage collection, centrifuge the pooled aspirate, and store the supernatant at negative 80 degrees Celsius. Resuspend the pellet in 200 microliters of ACK lysis buffer. After no more than two minutes at room temperature, dilute the lysis buffer with one milliliter of cold PBS and collect the cells by centrifugation.

Resuspend the pellet in the appropriate volume of PBS for the number of samples to be analyzed and aliquot the cells into the appropriate number of wells of a 96-well plate for the analysis. Collect the cells by another centrifugation, and resuspend the pellets in 50 microliters of FC block in PBS.

After 10 minutes at room temperature, add 50 microliters of the appropriate antibody cocktail of interest to each well, and incubate the cells for 30 minutes at four degrees Celsius protected from light. At the end of the incubation, centrifuge the plate and discard the supernatant, resuspending the pellets to a final volume of 200 microliters of FACS buffer per well for flow cytometric analysis.