Quantification of Candida Transcripts in Neutrophils

C. albicans cells were incubated with MPO^-/- mouse neutrophils in 8-well μ-Slide ibiTreat chambers as described above. After 90 min incubation, the supernatant was removed, and the remaining cells (neutrophils and ingested Candida) were scraped off the wells in lysis buffer of the RiboPure RNA purification kit for yeast (Ambion, ThermoFisher Scientific). Total RNA was extracted following the manufacturer’s instructions and concentrated using the RNA Clean & Concentrator-5 kit (ZymoResearch). cDNA was synthesized using SuperScript II Reverse Transcriptase (ThermoFisher Scientific) following the manufacturer’s instructions. Transcript quantification was conducted through real-time PCR analysis using SYBR green. The oligos used are listed in S3 Table. The experimentally validated TAF10 transcript [64 (link)] was used to normalize the qPCR data as we have done before [63 (link)].