Phrodo ifl green stp ester amine reactive

Cells from the human leukemia cell line, HL60 (ATCC), were labeled with pHrodo iFL Green STP Ester (amine reactive) (Thermo Fisher) as previously described (40 (link)), with minor modification. Briefly, HL60 cells were washed twice with PBS buffer and incubated at room temperature for 30 minutes with 0.1 μg/mL pHrodo. Cells were washed with PBS buffer twice and resuspended in RPMI medium containing 0.5% FCS at a density of 4.0 × 10⁵/mL to be used for engulfment. To 5.0 × 10⁴ BMDMs in 500 μL of RPMI medium containing 5% FCS in a 12-well plate, 2.0 × 10⁵ pHrodo-labeled HL60 cells were added and incubated at 37°C for 0, 30, 60, and 120 minutes. Cells were detached from the plate by trypsin and 1 mM EDTA and collected by centrifugation. Cells were labeled with surface markers CD11b and F4/80, then resuspended in 500 μL of PBS buffer containing 2% FCS and analyzed by flow cytometry with LSR II (BD Biosciences). Data were evaluated and analyzed with FlowJo. For time-lapse imaging, 1.5 × 10⁴/well BMDMs were passed onto an 8-well Lab-Tek II Chambered Coverglass (Nalge Nunc) and incubated at 37°C for 0, 30, 60, and 120 minutes with 6 × 10⁴ pHrodo-labeled HL60 cells in 250 μL of RPMI medium containing 5% FCS and observed by fluorescence microscopy.