Fura 2 am

For calcium imaging, we used the human tracheal epithelial cell line (HTEpC, C12644, PromoCell, Heidelberg, Germany). Cells were cultivated in an Airway Epithelial Cell Growth Medium Kit (C-21160) containing the Airway Epithelial Cell Growth Medium Supplement Pack (C-39160, PromoCell, Heidelberg, Germany). The airway epithelial cells were kept in a humidified chamber at 37 °C with air containing 5% CO₂. For the Ca²⁺ imaging, cells were seeded onto laminin-coated coverslips. Dye loading with 2.5 µM FURA-2 AM (Biotium, Fremont, CA, USA) in darkness was performed in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer for 30 min at 37 °C. For the contents of the HEPES buffer, see the section on drugs and buffer solutions. After the loading period, the cells were rinsed in fresh HEPES buffer and were transferred to the recording chamber of an upright fluorescence microscope equipped with 20 × immersion lenses (BX50 WI, Olympus, Hamburg, Germany), which contains 2 ml HEPES. The excitation light was provided by a 50 W xenon lamp. The microscope was equipped with a dichromatic excitation longpass mirror (400 nm).
The ratiometric dye, FURA-2 AM, was excited at 340 nm and 380 nm (± 8 nm) when equipped with bandpass excitation filters. The emitted fluorescence was directed through a dichromatic shortpass filter of 560 nm to a bandpass filter of 510 nm.

Claycomb cell culture medium was purchased from Sigma-Aldrich. FBS (fetal bovine serum), PBS (phosphate-buffered saline), HBSS (Hanks’ balanced salt solution), and penicillin/streptomycin antibiotic were purchased from Invitrogen/Thermofisher Scientific Pittsburgh, PA, USA. Other reagents used include BTP2 and ML204 (Millipore Sigma, St. Louis, MO, USA), EPI (Alfa Aesar, Haverhill, MA, USA), thapsigargin (TG, Adipogen, San Diego, CA, USA), fura-2 AM (Biotium 50033, Fremont, CA, USA), DAPI (Invitrogen D357, Carlsbad, CA, USA), and phalloidin (Enzo BML-T111, New York, NY, USA).

Reagents were as follows: ethylene glycol tetraacetic acid (EGTA) (Acros Organics, Geel, Belgium, 409910250), Fura-2 AM (Biotium, Kampenhout, Belgium, 50033), Annexin V-Fluorescein isothiocyanate (FITC) (Becton Dickinson, Franklin Lakes, NJ, USA, 556419), 7-aminoactinomycin D (7-AAD) (Becton Dickinson, 555815), U73122 (Enzo Life Sciences, Farmingdale, NY, USA, BML-ST391-0005), U73343 (Enzo Life Sciences, BML-ST392-0005), venetoclax (ChemieTek, Indianapolis, IN, USA, CT-A199), anti-human IgG/M (Jackson ImmunoResearch, West Grove, PA, USA, 109-006-127). The following antibodies were used: anti-IP₃R2 (Abiocode, Agoura Hills, CA, USA, R2872-3); anti-calnexin (Enzo Life Sciences, Farmingdale, NY, USA, ADI-SPA-865-D); anti-Bcl-2 (Santa Cruz Biotechnology, Dallas, TX, USA, sc7382HRP); anti-Bim (Bioké, Leiden, The Netherlands, 2819 S); anti-GAPDH (Sigma-Aldrich, St. Louis, MO, USA, G8795); anti-vinculin (Sigma-Aldrich, Munich, Germany, V9131). The sequences of the peptides used in this study were: BIRD-2 (RKKRRQRRRGGNVYTEIKCNSLLPLAAIVRV) and TAT-Ctrl (RKKRRQRRRGGSIELDDPRPR). These peptides were synthesized by LifeTein (South Plainfield, New Jersey, USA) with a purity of at least 85%. The IP₃ sponge (pEF-GSTm49-IRES-GFP) is a protein constructed from the IP₃-binding core of the type 1 IP₃R with a single amino acid substitution (R441Q) that has a very high affinity for IP₃ [29 (link)].

Cells were stained with Fura-2 AM (3 µM) and 0.01% pluronic F-127 (both from Biotium Inc., Fremont, CA, USA) for about 1 h. Coverslips were mounted on an inverse microscope. The extracellular solution contained (in mM): NaCl 145, KCl 5, CaCl₂ 1.25, MgCl₂ 1, Glucose 10, Hepes 10). For calcium-free experiments, CaCl2 was replaced by 5 mM EGTA. Solutions were applied using a gravity-driven superfusion system. Fura-2 was excited using a microscope light source and an LEP filter wheel (Ludl electronic producs Ltd., Hathorne, CA, USA) to switch between 340 and 380 nm. Images were exposed for 20 and 10 ms, respectively, and acquired at a rate of 1 Hz with a CCD camera (Cool SNAP EZ, Photometrics). Data were recorded using VisiView 2.1.1 software (all from Visitron Systems GmbH, Puchheim, Germany). Background fluorescence was subtracted before calculation of ratios. A 60 mM potassium chloride stimulus was applied as a control at the end of each experiment for DRG neurons and 5 µM ionomycin was used in HEK293t cells. Cells which did not respond to potassium/ionomycin, or showed no functional expression of TRPV1 or TRPA1 in experiments on HEK293t cells, were excluded from the analysis. Averaged results are reported as means (± S.E.M.).

MDMs (day 8) were detached, counted and adjusted to a density of 1 × 10⁷ cells/ml, and labeled with 840 nM CFDA-SE (Life Technologies) in PBS for 15 min at 37 °C. Labeled cells were washed and quenched in medium for 30 min at 37 °C, 5% CO₂ then seeded onto poly-d-lysine (Sigma-Aldrich) coated glass coverslips (2 × 10⁵ cells/coverslip). After 24 h cells were left untreated or pulsed with 1 µg/ml sAgs for 2 h at 37 °C, 5% CO_2, as indicated. Concurrently, autologous CD4⁺ T cells were labelled in medium at 37 °C for 30 min with a ratiometric calcium sensitive dye (5 µM fura-2/AM; Biotium Inc), then resuspended at 1 × 10⁶ cells/ml in phenol-red free media and incubated at RT for 30 min. MDMs on coverslips were washed prior to being affixed into an RC-21BRFS closed perfusion chamber (Warner Instruments) using vacuum grease (Dow Corning). The device was filled with phenol-red free medium and sealed by applying another coverslip to form the upper face of the chamber. Imaging was performed with an incubated inverted epifluorescence microscope (see supplemental material). Experiments were repeated with three technical replicates on cells from N ≥ 3 donors.