β mercaptoethanol

Casein micelles (CSMs, 92% protein) were purchased from VWR Chemicals (Leuven, Belgium). Fluorescein isothiocyanate was obtained from Fluorochem Ltd. (Hadfield, UK). β-mercaptoethanol, sodium hydroxide, and sodium chloride were acquired from AppliChem GmbH (Darmstadt, Germany). Sodium dodecyl sulfate (SDS) and Coomassie blue dye (G250) were acquired from Sigma Aldrich (St. Louis, MO, USA). 8-Anilino-1-naphthalenesulfonic acid (ANS) was purchased from Cayman Chemical Company (Ann Arbor, Michigan, USA). Hydrochloric acid, phosphoric acid, methanol, and ethanol absolute were obtained from Merck (Darmstadt, Germany).

Total RNA (i.e., both bacterial and plant RNA) was extracted from all collected leaves. Owing to the richness in polysaccharides and phenolic compounds of strawberry plant tissues, the extraction was performed with a modified method of Christou et al. [45 (link)], as outlined below. Collected leaves were cut into three sections, used as triplicates of 100 mg initial material and extracted in parallel. The extraction buffer (EB) was supplemented with freshly added 2% β-mercaptoethanol (Applichem GmbH, Darmstadt, Germany) in order to preserve samples from RNase activity; the powdered leaves were transferred in ice-cold EB and let on ice for 15 min with shaking every 3 min, in order to allow the extraction buffer to access all plant material and avoid sedimentation of material, instead of directly adding phenol/chloroform/isoamyl alcohol (25:24:1 v/v; AppliChem GmbH, Darmstadt, Germany); RNA samples were washed twice with 70% (v/v) ethanol in order to remove traces of phenols and other potentially interfering components; and nucleic acid pellet was air-dried at room temperature for 2 min and subsequently dissolved in 30 µL RNase free water on ice for 15 min.

After 48 hours of activation, T cells were pelleted and washed once with ice-cold PBS, before the cell pellet was treated with the lysis buffer provided in a NucleoSpin RNA Isolation Kit (Macherey-Nagel, Dueren, Germany) supplemented with β-mercaptoethanol (AppliChem GmbH) as indicated in the kit manual and either stored in a -80°C freezer or directly used for RNA isolation. Total RNA was isolated from the cell lysate using the NucleoSpin RNA Isolation Kit according to the manufacturer’s instructions. RNA concentrations were determined with an Infinite 200 PRO Microplate Reader (Tecan Group Ltd., Mannedorf Switzerland) and NanoQuant Plate™ (Atlantic Lab Equipment, Beverly, MA, U.S.A.).

Cells were isolated as described above from inguinal, abdominal and thoracic mammary glands. Following isolation, cells were resuspended in full medium (RPMI (Seraglob), 10% FBS (Gibco), 0.01 M HEPES (Gibco), 1 mM sodium pyruvate (Gibco), 2 mM Glutamax (Gibco), 1% Penicillin–Streptomycin (Gibco), 1% nonessential amino acids (Gibco), 57.2 μM β-mercaptoethanol (AppliChem)) with GolgiPlug and GolgiStop 1:1,000 (BD) and exposed to Zymosan 50 µg ml^–1 (InvivoGen) or LPS 300 ng ml^–1 (Sigma) or left unstimulated (negative control). The cells were incubated for 6 h at 37°C, washed and stained for surface markers as described above, then fixed with Cytofix/Cytoperm (BD Biosciences) for 20 min at 4°C, washed with Perm buffer (2% BSA, 0.5% Saponin, 0.0002% sodium azide in PBS), stained with intracellular antibody mix in Perm buffer for 25 min (TNF (clone MP6-XT22 1:400) and proIL-1β (clone NJTEN3 1:200), purchased either from eBioscience or Biolegend), washed with Perm buffer, resuspended in PBS and acquired.

For mRNA expression analyses, CD8⁺ T cells upon the indicated treatment were washed once with ice-cold PBS, before adding the lysis buffer from the NucleoSpin RNA kit (Macherey–Nagel, Düren, Germany) supplemented with β-mercaptoethanol (AppliChem GmbH). Total RNA was isolated according to the company’s protocol and then subjected to quantitative PCR (qPCR). For each sample, cDNA was synthesized from 500 ng of total RNA using a RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific). qPCR reactions were done using 2X SYBR Green qPCR Master Mix (Promega, Madison, WI, USA) according to the manufacturer’s recommendations using the primers listed in Additional file 1: Table S1. qPCR data are presented as ΔΔCq (fold change) calculated using the TATA-binding box protein (TBP) and succinyldehydrogenase subunit A (SDHA) as housekeepers according to the following formula: $\mathrm{\Delta }\mathrm{\Delta }Cq=\left(C,q,H,K,-,C,q,G,O,I\right)treated-\left(C,q,H,K,-,C,q,G,O,I\right)untreated$ [48 ]. CqHK represents the mean of the quantification cycle (Cq) values for the housekeepers (HK) and the CqGOI is the Cq of the gene of interest (GOI) in the indicated condition.