A specific and sensitive method for detection of hypochlorous acid for the imaging of microbe-induced HOCl production

Abstract

A specific and sensitive fluorescence-based method was developed for the imaging of microbe-induced HOCl production. Furthermore, we demonstrate dual oxidase (DUOX)-mediated HOCl generation in the mucosa of live animals providing a novel insight into mucosal innate immunity.

Methods to study Drosophila immunity

“…This probe (R19S) is a rhodamine-based sensor which specifically reacts with HOCl and not with other ROS [102]. In the same publication, the group utilized 100 mM of Amplex UltraRed reagent (Invitrogen) and 0.2 U/ml of horseradish peroxidase (Sigma) reaction buffer to measure H 2 O 2 in Caenorhabditis elegans.…”

The RclR Protein Is a Reactive Chlorine-specific Transcription Factor in Escherichia coli

“…HOCl Survival Assays-HOCl survival assays were performed as described previously (20). -TAC TAA GAT TGT TGG TGT TTG TAA TCA AAA ACC ACT CAG GAG TCT GAT ATG GTG TAG GCT GGA GCT GCT TC-3Ј [2] 5Ј-AAC TCC CGG AAT GCG GAT TAT CTT TGC CCT GAT TTC TGG CGG TTT TAT TCA CAT ATG AAT ATC CTC CTT AG-3Ј [3] 5Ј-CGT CTA TAG TCA TGA TGT CAA ATG AAC GCG TTT CGA CAG GAA ATC ATC ATG GTG TAG GCT GGA GCT GCT TC-3Ј [4] 5Ј-CTT TTC TCT GAG ACG CCA GAA TAT TTG TTC TGG CGT CTG ATT TTG AGT TTA CAT ATG AAT ATC CTC CTT AG-3Ј [5] 5Ј-TCT CTG TTA CTC TAT AAC TTC TTA ATC ACT TCA TTG ATG GTA TTT TAT ATG GTG TAG GCT GGA GCT GCT TC-3Ј [6] 5Ј-TAT CTC CCC GAC TTA ACA GGT GCA GGT ATT TTT CCA TTG TGA ACA TCC TTA CAT ATG AAT ATC CTC CTT AG-3Ј [7] 5Ј-TAA TAA TTC AAC CGC AAC AGC AGT ATT GTA TAA ATA AGG ATG TTC ACA ATG GTG TAG GCT GGA GCT GCT TC-3Ј [8] 5Ј-AGG GGG CCT GAC GCC TGA AAA AGT GAA CAA CAG ACA GTG TTC GGA TTA TCA CAT ATG AAT ATC CTC CTT AG-3Ј [9] 5Ј-TTC GAA TTC AGG AGT CTG ATA TGG ATG CCC-3Ј [10] 5Ј-CTT AAG CTT TCA GGG TGC AAG CTG TCT GA-3Ј [11] 5Ј-GAA CGA TCG ATA AGA ATG CGG TGT TAG GAA GTG ACT GG-3Ј [12] 5Ј-CGA CCT GTA TTG TCG CGG GCA CTC TTC GGT TG-3Ј [13] 5Ј-GAA TTA TCG GTT ATT CGT TGG GCG GCG TTA ACG CAA GGA GCG GC-3Ј [14] 5Ј-CCA CAA AAT TCA GCT GCG CGC CTG AGT CAT GTC G-3Ј [15] 5Ј-CAC GAT TGA TAA AAA CGC GGT CCT GGG TTC TGA CTG-3Ј [16] 5Ј-GAA TCC ACC TGC ATT GTT GCG GGT ACG CTG CGT CTG CAA C-3Ј [17] 5Ј-ACT GAG CGT TAT TCG TTG GGC GGC ACT GAC CCA GGG TGC A-3Ј [18] 5Ј-AGA ATA GTG CGG CGC GCC TGT CCC ACG TGG ATA A-3Ј [19] 5Ј-AAG AAC TTA CCT GGT CTT GAC ATC-3Ј [20] 5Ј-CAG TTT ATC ACT GGC AGT CTC CTT-3Ј [21] 5Ј-AAA AAC ATT CTC AGT TTG CTT CTG C-3Ј [22] 5Ј-CGC TAT TTG TAG ACG CAA CTT TGT T-3Ј [23] 5Ј-AGG GTT GGT ATG ACA GAA GAA CAA G-3Ј [24] 5Ј-TAT ATT GAT CAT CTC GTG GGA GTC A-3Ј [25] 5Ј-CAC TAT TAT CTG GCG TTA TGG CAT T-3Ј [26] 5Ј-TAT ACA ATA CTG CTG TTG CGG TTG-3Ј [27] 5Ј-TCT TTT ATG AAC ACC CGG AAG ACT A-3Ј [28] 5Ј-ATC AGT CCT CCC AAT AAA CCT AAC C-3Ј [29] 5Ј-GGT CCT TCT ATC ACT ACT TTC GCT CT-3Ј …”

“…Plasmid Construction-The rclR coding sequence plus 11 bp of 5Ј-sequence was amplified from E. coli MG1655 genomic DNA with primers [9] and [10] (Table 1) and cloned into the EcoRI and HindIII sites of plasmid pBAD30 (36) to yield plasmid pRCLR1. The QuikChange site-directed mutagenesis kit (Stratagene), modified to use only a single primer and 35 cycles of amplification, was used to mutate pRCLR1 with primers [11], [12], [13], or [14] to yield plasmids pRCLR8, pRCLR9, pRCLR14, and pRCLR15, containing rclR alleles encoding RclR C21A , RclR C89A , RclR H42A , and RclR H75A , respectively.…”

The Fluorescein-derived Dye Aminophenyl Fluorescein Is a Suitable Tool to Detect Hypobromous Acid (HOBr)-producing Activity in Eosinophils

“…The rhodamine-based probe R19-S was also reported to specifically detect HOCl (21). Again both dyes, HySO x and R19-S, have not yet been applied on HOBr.…”