Percoll gradient

C. tyrobutyricum UC7086 from Università Cattolica Culture Collection (Piacenza, Cremona, Italy) was used for the development of the LAMP assay and for the milk artificial contamination to evaluate the performance of the method. C. tyrobutyricum spores were obtained using the protocol described by Bassi et al. [21 (link)]. Briefly, the strain was cultured in reinforced clostridial medium broth (RCM) (Oxoid, Altrincham, UK) and incubated at 37 °C in anaerobiosis. 1% of well-grown suspension was inoculated into a regenerated cellulose tubular membrane located in a bottle with 400 mL of RCM broth. After five days of anaerobiosis at 37 °C and at least 15 days of aerobiosis, the membrane content was checked to verify the presence of a high percentage of spores by visualization with phase-contrast microscopy (Nikon, Tokio, Japan). Spores were then collected, washed several times with sterile distilled water, and purified using Percoll gradient (Merck KGaA, Darmstadt, Germany) as previously described [43 (link)]. No vegetative cells were observed at the phase-contrast microscopy. Purified spores were stocked at −20 °C. The spore counts were determined by plating 10-fold dilutions of the obtained spore solution after pasteurization for 10 min at 80 °C. The stock was used to develop the LAMP assay and to artificially inoculate milk samples.

Pea and maize seedlings were grown in a darkened growth chamber at a constant temperature of 20 °C for 7 and 13 days, respectively. Etioplasts were isolated according to Blomqvist et al.^{19 (link)} with minor modifications. Briefly, leaves were ground with a Waring blender in ice-cold buffer A (pea: 50 mM Tricine/NaOH pH 7.5, 300 mM sorbitol, 10 mM CaCl₂, 10 mM MgCl₂; maize: 20 mM HEPES/NaOH pH 8.0, 300 mM sorbitol, 10 mM NaHCO₃, 5 mM MgCl₂), then filtered through four layers of Miracloth (Merck). The organelles were pelleted (4,500g, 15 min, 4 °C), resuspended and further purified by centrifugation (4,500g, 20 min, 4 °C) on a discontinuous 40–80% Percoll gradient (Merck) in buffer A. Intact etioplasts were isolated from the 40–80% interface and washed twice in buffer A to eliminate residual Percoll. All steps were carried out in the dark or under safe green light.

When required, P. falciparum cultures were synchronized to the ring stage prior to enrichment using one round of 5% D-sorbitol (Sigma Aldrich, St Louis, MO)^{51 (link)}. SLO lysis was performed as previously described but with modifications^{24 (link)} (Fig. 1b; Supplementary Method S1). Briefly, erythrocyte density was measured using a Cellometer Auto T4 (Nexcelom Biosciences, Lawrence, MA). Cell density was adjusted to 2 × 10⁹ erythrocytes/mL. The desired amount of SLO (between 0U and 55U) was added in a ratio of 2 parts SLO solution to 5 parts erythrocytes. Samples were mixed well by pipetting and incubated at room temperature for precisely 6 min. Five-10 volumes of 1X PBS or non-cholesterol containing media (ex. RPMI 1640 HEPES) were added and cells were centrifuged at 2,500 × g for 3 min. After removal of the supernatant, cells were washed twice more with 1X PBS or non-cholesterol containing media. Following SLO lysis, cells suspended in 1X PBS were layered onto a 60% Percoll gradient (Sigma Aldrich, St Louis, MO) and centrifuged at 1,500 × g for 5–10 min depending on the volume of the gradient. The top layer of Percoll was discarded while the lower cell pellet was collected and washed twice with 1X PBS or media.

Lymphocytes were isolated from tissues as previously described (Masopust et al., 2001 (link)), with some modifications. Briefly, bronchoalveolar lavage (BAL) of the airways was performed with 1 ml of PBS containing 1% BSA prior to perfusion of the lungs with PBS. Lungs were treated with 1 mg/ml Collagenase plus 1 mg/ml DNase (Sigma-Aldrich) in 3 ml of RPMI for 45 min at 37° C. Single cell suspensions were obtained by pushing digested lungs, spleens, and lymph nodes through 40 μM mesh screens (BD Biosciences). Lung lymphocytes were purified by centrifuging (2000 rpm, 4°, for 20 min) on a 44/67% Percoll gradient (Sigma-Aldrich).