Table 3.

Burkholderia cenocepacia and Ubiquitin Staining for High-Content Imaging

Step	Parameter	Value	Description
1	Differentiation	48 h	Human THP1 monocytes were differentiated to macrophages by treating with 50 ng/mL PMA and grown in RPMI media with 10% heat-inactivated FBS
2	Rapamycin treatment	50 μg/mL	If required for experimental protocol, treat cells with rapamycin for 2 h before infection
3	Infect cells	1 h	Burkholderia cenocepacia J2315 was added to cells in fresh media (no rapamycin) at an MOI of 1 (5 × 105 bacteria/mL)
4	Combination antibiotic treatment	2 h	Infected media was aspirated and replaced with media treated with 250 μg/mL gentamicin and 500 μg/mL ceftazidime
5	Media replacement	3–21 h	Antibiotic media was replaced with nonantibiotic RPMI for the remainder of the time course
6	Fixation	3×, 10 min	Cells were washed thrice in PBS and fixed with 4% PFA
7	Permeabilize cells	10 min	Cells were washed once in PBS and permeabilized with Triton X-100 0.1%
8	Block cells	1 h	Cells were washed twice in PBS and blocked in 5% BSA-PBS
9	Primary and secondary antibody staining; nuclear staining		As described in Table 1, steps 8–14

FBS, fetal bovine serum; PMA, phorbol 12-myristate 13-acetate; MOI, multiplicity of infection.