Goat anti rabbit alexa488 antibody

Neutrophil extracellular trap visualization was performed as described previously (29 (link)). Briefly, 5 × 10⁶ neutrophils were infected with GAS WT and ΔSLO mutant was added at MOI = 1 and incubated for 4 h in 37°C/5% CO₂. Cells were fixed with 4% paraformaldehyde and stained with anti-myeloperoxidase (MPO) antibody (1:300 diluted, Calbiochem) in PBS + 2% bovine serum albumin (BSA, Sigma) for 1 h at RT, followed by incubation with goat anti-rabbit Alexa 488 antibody (1:500 dilution, Life Technologies). Cells were counterstained with ProlongGold + 4′,6′-diamidino-2-phenylindole (DAPI, Invitrogen) and imaged on a fluorescent microscope. Representative, randomized images (n = 5) were taken for each condition and individual experiment. The ratio of NET-releasing cells to non-NET-releasing cells was determined as % of total cells releasing NETs.

Raw-Blue cells were transfected or infected as indicated above and harvested at 12hpi into 70% ethanol. The fixed cells were permeabilised with PBS buffer containing 0.1% saponin and 0.1% bovine serum albumin. Cells were incubated with rabbit anti-TLR antibodies (TLR7 (Abcam 45371) and TLR8 (Abcam 180610); 1/50 dilution) for 20 min, washed and labeled with goat anti-rabbit Alexa488 antibody (Invitrogen) for a further 20 min. Labeled cells were washed thoroughly before being analysed using BD Fortessa. Each sample contained 20,000 gated cells for analysis using FlowJo 10.1.

Cell viability was assessed on 96-well plate using WST-1 colorimetric assay according to the manufacturer’s instructions [36 (link)] (Roche). TUNEL assay using Apodirect TUNEL assay kit (Millipore) for cell apoptosis was performed on a flow cytometer (FACSCanto, Becton Dickinson) using channel FL1. Also, to analyze whether the cell death was apoptosis the cells (5 × 10⁶) were incubated with cleaved caspase 3 antibody (cell signaling Technology) at a ratio of 1: 100 for 1 h at 37°C followed by secondary goat anti-rabbit alexa 488 antibody (Invitrogen) (1: 1000) for 30 min at 37°C. Finally, the samples were washed 3 times followed by flow cytometry at FL1 channel.

For immunofluorescence of Drosophila wing discs, the crosses were raised at 29°C (MS1096‐GAL4; UAS‐GFP × UAS‐GFP as control or x UAS‐dPANK/fbl‐RNAi) or 22°C (MS1096‐GAL4; UAS‐GFP × UAS‐GFP as control or x UAS‐mtacp‐RNAi #29528). Wandering L3 larvae (day 5) were collected and their wing discs dissected in ice‐cold phosphate‐buffered saline (PBS). The discs were fixed with 4% formaldehyde (Thermo Scientific Pierce) for 30 min, washed for 3 × 20 min with phosphate‐buffered saline (PBS) + 0.1% Triton X‐100 (Sigma Aldrich), and afterward incubated in primary antibody rabbit anti‐Fbl (Bosveld et al, 2008; 1:500) or rabbit anti‐mtACP (ThermoFisher PA5‐22191, 1:500) in PBS + 0.1% Triton X‐100 overnight to visualize the presence/absence/localization of Fbl or mtACP. After an additional washing step of 3 × 20 min in PBS + 0.1% Triton X‐100, the discs were incubated in secondary goat anti‐rabbit‐Alexa488 antibody (Molecular Probes) for 2 h at room temperature. DAPI (0.2 μg/ml; Thermo Scientific) was used to visualize DNA. Finally, the samples were mounted in 80% glycerol and analyzed using a Zeiss‐LSM780 NLO confocal microscope with Zeiss software. Adobe Photoshop and Illustrator (Adobe Systems Incorporated, San Jose, California, USA) were used for image assembly. A complete list of antibodies used can be found in Appendix Table S3.

Laid embryos were collected as mentioned above. The stage of embryos was staged as described42 (link) and doubly checked by WT or WT-like control embryos (the siblings) that were collected in the same time windows. Embryos were dechorionated and sorted into different dishes of mibⁿⁿ²⁰⁰² homozygotes and WT siblings at 14 hpf. WT and mibⁿⁿ²⁰⁰² homozygotes that did not exposed to UV were moved to room temperate for 15 min. At the same time, the WT-UV control was placed in laminar flow hood with UV on for 15 min. Afterwards, all dishes were moved to 28.5 °C incubator for 1 hour before collected. Collected embryos were dehydrated by −20 °C methanol/acetone (1:1) and stored at −20 °C. Embryos were then sorted into 10 embryos/vial and washed by 1% Triton/PBS several times on shaker. After overnight blocking, embryos were washed by 2% BSA/1% Triton/PBS at 4 °C on shaker, and 1:1000 rabbit anti-human γ-H2AX (Cell signaling, #9718) in 2% BSA/1% Triton/PBS was used for overnight reaction. After intensive wash, 1:1000 goat anti-rabbit Alexa-488 antibody (Molecular probe) in 2% BSA/1% Triton/PBS was used for overnight reaction. After intensive wash, embryos were fixed by 4% paraformaldehyde. Embryos were further deyolked and flat-mounted in 75% glycerol to avoid strong background fluorescence from yolk. Images were taken by Zeiss Axiovision Imager A1.

Immunofluorescence analysis on Drosophila S2 cells was performed as described in detail previously²⁹ . Briefly, the cells were stained with rabbit anti-AcLys antibody (Cell Signaling Cat No: 9441, 1:500) as primary and goat anti-rabbit-Alexa488 antibody (Molecular Probes) as secondary antibody. Rhodamin-Phalloidin (20U/ml) (Invitrogen) was used to detect F-actin, and DNA was detected by DAPI (0.2 μg/ml) (Thermo Scientific). The samples were analyzed with a Leica fluorescence microscope with Leica software. Adobe Photoshop and Illustrator (Adobe Systems Incorporated, San Jose, California, USA) were used for image assembly.