8 chamber μ slide

MEFs were seeded at 150,000 cells/well of a 6 well plate. The next day cells were washed with PBS and media without antibiotics was added. Cells were transfected with siRNA’s complexed with Lipofectamine RNAiMAX according to manufacturers instructions. Briefly, 7.5 µL of 20 µM stock stealth siRNA oligo was added to 150 µL of optimem. Nine microlitre of RNAiMAX was added to another 150 µL of optimum. The tubes were mixed together before incubating at room temperature for 5 min. 250 µL of this was then added to the cells and incubated overnight. Cells were seeded at 50,000 cells/well of an 8 chamber μ-slide (Ibidi) and allowed to plate down overnight before microscopy was performed as described above. The following stealth siRNAs were used from Thermo Fisher Scientific; cIAP1 (MSS273215), cIAP2 (MSS202113) and XIAP (MSS202115). Activity of each siRNA was confirmed using qPCR with SYBR Select Master Mix (applied biosystems # 4472908) and the following primers: cIAP1 (Forward 5′-GAAGAAAATGCTGACCCTACAGA-3′, Reverse 5′-CATGACGACATCTTCCGA-3′), cIAP2 (Forward 5′- TCGATGCAGAAGACGAGA-3′ Reverse 5′-TTTGTTCTTCCGGATTAGTGC-3′, XIAP (Forward 5′-GCTTGCAAGAGCTGGATTTT-3′, Reverse 5′-TGGCTTCCAATCCGTGAG-3′). Actin was used as a reference gene. PCR was done in 384 well plates in a 7900HT Fast Real-Time PCR System.

Non-adherent K562 cells were counted and collected in 1.5 ml Eppendorf tubes at 30 000 cells per tube. After a wash, cells were re-suspended in 200 μl of phenol-red free media containing 5% FCS and fluorescein-labeled peptides (c(peptide) = 1 μM, t = 30 min) in the Eppendorf tubes at 37°C with vertical rotation to avoid cell pelleting. After incubation, cells were washed once with phenol-red free media, re-suspended again in the same media, and transferred into the wells of an 8-chamber μ-slide (Ibidi). Cells were immediately imaged at 37°C with an SP5 Laser Scanning Confocal Microscope (Leica). Fluorescein was excited with an argon ion laser at wavelength of 488 nm and fluorescence was detected in the range 505–530 nm.

Cells seeded in an 8-chamber μ-slide (Ibidi, Verona, WI, USA) were primed with 1 μg/ml E. coli O111:B4 LPS (Sigma-Aldrich, St. Louis, MO, USA) for 2–4 h followed by the addition of inhibitors or other treatment as indicated, then stimulation with 1–5 mM ATP (Sigma-Aldrich). Alternately, cells were stimulated by addition of 20 μM nigericin (#11437, Cayman Chemical, Ann Arbor, MI, USA). Samples were imaged on a Nikon Ti microscope equipped with a C2si confocal scanner (Nikon Instruments, Melville, NY, USA) and Tokai Hit stage-top incubator (Tokai Hit Co., Shizuoka, Japan). Excitation laser lines were 408, 488, 561 and 639 nm and emission was collected by photomultipliers filtered for the standard DAPI, FITC, TRITC and Cy5 bandwidths. Objectives used were × 20 air 0.75 NA, × 60 oil immersion 1.4 NA or × 60 water immersion 1.2 NA, all from Nikon. Where indicated, cells were imaged in the presence of 5 μM TO-PRO-3 (Molecular Probes). For calcium imaging, cells were loaded with 1 × Fluo-4 DIRECT solution (Molecular Probes) and incubated for 30–60 min before imaging. For analysis of mitochondrial ROS production, MitoSOX red (Molecular Probes) was added 15 min after stimulation with ATP.