determine cell titer. The required number of bacteria for 
an MOI of 20:1 was taken from this culture, and washed three times with Middlebrook 7H9 media supplemented with 0.05% Tween-80, then resuspended in 200 mL of Middlebrook 7H9 with Tween-80. A final concentration of 30 mg/mL of Alexafluor 633 succinimidyl ester was added, and the mixture was purchase Actimid incubated at 37uC for 1 hour with rotation. Bacteria were centrifuged at 130006g for 1 minute. PI3K p110a and Phagosome Maturation For phagocytosis assays, bacteria were then washed three times with RPMI, resuspended to give a final cell titer of 1.6E6/mL and used to infect PMA-differentiated THP-1 in culture plates. Phagocytosis was synchronized by placing cells at 4uC for 20 minutes prior to incubation at 37uC /5% CO2. Timepoints were taken by washing cells three times with PBS, resuspending cells in 1 mL of PBS with 0.2% trypan blue, then pelleting the cells by centrifugation at 15006g for 2 minutes. Cell pellets were washed three times with PBS, then resuspended and fixed in 2% paraformaldehyde in PBS and stored at 4uC. For quantification of bacteria uptake, samples were washed once, resuspended in PBS, and run on a flow cytometer. Data were analyzed using FlowJo software. 3 hours at room temperature. Phagosomes were then washed three times with PBS PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22212565 containing 0.05% Tween-20, and incubated with secondary antibody in blocking buffer for 1 hour at room temperature. After washing three times with PBS-T, phagosomes were resuspended in PBS, then analysed on a FacsCalibur flow cytometer. The resulting data was analyzed with FlowJo software. Confocal Microscopy Control and p110a knockdown cells were seeded onto coverslips and differentiated overnight with 10 ng/mL PMA. Cells were then washed once with complete medium and allowed to rest for 4 hours prior to bead treatment as described above. Cells were allowed to ingest beads for 15 min after which they were washed three times with warm PBS and replenished with complete medium. After 2 h of incubation, cells were fixed with 2% paraformaldehyde, washed three times with PBS, then permeabilized with 0.1% Triton X-100 in PBS at room temperature for 10 min. Following washes with PBS, permeabilized cells were incubated with LAMP-1 antibodies overnight at 4uC, followed by Alexafluor 555 – conjugated anti-mouse secondary for 1 h at room temperature. After washing with PBS, coverslips were mounted onto Prolong Gold Antifade reagent with added DAPI stain, and Z-stacks of cells were imaged using a Leica DMIRE2 inverted microscope equipped with a SP2 AOBS laser scanning head. Images were processed using the MacBiophotonics version of the Image J software. For Texas Red dextran experiments, cells were seeded onto coverslips as described, with the addition of 0.25 mg/mL of Texas Red dextran to the culture medium. Cells were then incubated overnight, washed three times with warm PBS and given fresh medium. This was followed by a 4 h incubation in order to chase fluorescent dextran into the lysosomal compartment. Cells were then fed beads, allowed to phagocytose for 15 min, washed, given fresh medium, and incubated for 2 h prior to fixation. At least 100 phagosomes were counted from three independent experiments for each set of experiments. BSA-coupling and Alexafluor 633 Labelling of Magnetic Beads and Bead-treatment of THP-1 Cells Carboxylic acid functionalized 3 mm diameter magnetic beads were coupled with bovine serum albumin as per manufacturer’s protocol.
