Movie 1

Monitoring intra-phagosomal Ca²⁺ changes in mouse bone marrow derived macrophage from Trpm2^+/+ mice. Cellular and intra-phagosomal Ca²⁺ was monitored with fluo3 as Ca²⁺ indicator, and phagosomal acidification was monitored using pH sensitive fluorescent dye pHrodoRed conjugated to Staphylococcus aureus (SA). BMDMФs from Trpm2^+/+ were first loaded with 5 µΜ fluo3-AM (membrane permeant) for 30min at 37^oC and cells were then infected with SA conjugated with the pH-sensitive fluorescent dye pHrodo with a MOI of 10. Dye emission was determined by a Zeiss LSM 710 confocal microscope using a 488 nm laser and a 561 nm laser, a 63X objective lens (NA=1.3), and an emission bandwidth of 500-535 nm and 600-680 mm. Images were collected every 10s with LCS software and were processed and analyzed with Fiji software. Note the rapid appearance of the red fluorescence from pHrodo used as a marker for both phagososome and phagosomal acidification, but the green fluorescence from fluo3 (Ca²⁺ signal) did not increase with time in the corresponding phagosomes. The merged video clearly shows the strong red color in phagsosomes due to reduced phagosomal Ca²⁺ accumulation (green).