Snap surface alexa fluor 488

The AF488 assay was adapted from reference^{9 (link)} with some modifications. SNAP-Surface Alexa Fluor 488 (NEB) was dissolved in DMSO for 5 mg/ml stock solution and stored at −80 °C for up to several weeks. 2 ml of the strain to be tested was cultured in 7H9 medium until exponential growth phase with optical density (OD_600nm = 0.6–0.8). 1 ml bacteria were inoculated in 7H9 medium with and without 1 μg/ml rifampicin respectively for 3 hours. After two washes with1ml PBST, bacteria was re-suspended in 50 μl diluted AF488 (working concentration 200 μg/ml). After incubating in the dark at room temperature for 5 min, bacteria were transferred into a fresh tube and then washed twice more with 1 ml PBST. Bacterial pellets were re-suspended in 500 μl 7H9 fresh medium. 100 μl of the suspension were inoculated into 7H9 medium containing 0 μg/ml, 10 μg/ml, 50 μg/ml rifampicin. After 16 hours culture in 37 °C shaking incubator in the dark, 100ul samples were fixed by 100ul 4% Paraformaldehyde (PFA) at room temperature for 20 min. BD Accuri C6 desktop flow cytometry was used to collect fluorescence intensity with 488 nm excitation laser and 533 nm emission filter. % Dim cells representing the relative percentage of growing cells were analyzed by Flow-Jo software.

HEK293 cells transiently transfected with SNAP-tagged FZD constructs and mG constructs or stably expressing SNAP-FZD₆ and SNAP-FZD₆ R416A^6.32 were grown in a 6-well plate. On the day of the experiment, the cells were detached with ice-cold 10 mM EDTA/PBS and then centrifuged at 400× g for 5 min in complete DMEM medium. The cells were resuspended in ice-cold 0.5% BSA/PBS, counted and transferred (3 × 10⁵ cells) to a round-bottom 96-well plate. The plate was then centrifuged at 400× g for 5 min and subsequently cells were incubated with SNAP-substrate: either SNAP-Surface Alexa Fluor 488 (NEB #S9129S), SNAP-Surface Alexa Fluor 647 (NEB #S9136S) or SNAP-Cell 647-SiR (NEB #S9102S) at 1:200 dilution in complete DMEM medium for 30 min at 37 °C. The plate was centrifuged twice, cells were resuspended in ice-cold 0.5% BSA/PBS, and assayed immediately on an ADP Cyan flow cytometer. The median fluorescence intensity (MFI) data were analyzed using FlowJo V10 (Tree Star).

All reagents unless otherwise stated were purchased from Sigma-Aldrich. Xba1 and Xho1 restriction enzymes, pNLF and FuGENE HD transfection reagent, pNLF vectors, NanoGlo Substrate, AF488 HaloTag Ligand and HaloTag NanoBRET 618 Ligand were purchased from Promega. OptiMEM and NHS Ester linked 5 and 6-Carboxytetramethylrhodamine (TAMRA) mixed isomers were purchased from Thermo-Fisher Scientific. SNAP-Surface Alexa Fluor 488 was purchased from New England BioLabs. An IL23R-MYCDDK expression plasmid (accession code NM_144701) and an IL12Rβ1 expression plasmid (accession code NM_005535) were purchased from Origene. Recombinant NanoLuciferase expressed in E. coli and purified using an N terminal His-tag was gifted by Bradley Hoare of the Veprintsev lab, University of Nottingham. Rabbit anti-Nanoluciferase was kindly gifted by Promega. Recombinant IL-23 protein was gifted by Surjit Bains at GlaxoSmithKline (Stevenage, UK). The protein was originally purified through expression in HEK293-F cells via co-transduction with BacMam viruses containing the p19-His and p40 transcripts. Heterodimeric IL-23 was then purified from the supernatant using a Ni-Sepharose column.

SNAP-tagged proteins were labeled with SNAP-Surface Alexa Fluor 488, SNAP-Surface Alexa Fluor 546, and SNAP-Surface Alexa Fluor 647 (New England Biolabs). Protein (5 µM) and dye (10 µM) were mixed and allowed to react in 50KMEH5Gd at RT for 2 hr. Labeled proteins were desalted into 50KMEH5Gd buffer and concentrated. Extinction coefficients of fluorophores were calculated from a standard curve and are as follows: Alexa 488 at 495 nm, 95,000 M^–1*cm^–1; Alexa 546 at 556 nm, 120,000 M^–1*cm^–1; Alexa 647 at 650 nm, 255,000 M^–1*cm^–1. Protein labeling efficiency was calculated by dividing protein concentration by dye concentration—for Alexa Fluor 488 the labeling efficiency was estimated at ~100%, for Alexa Fluor 546 the labeling efficiency was estimated at ~60%.