6 well polystyrene plates

For Western blots, cells were transfected on 6-well polystyrene plates (Corning). 24 h after transfection, the cells were washed twice with ice-cold PBS followed by scraping and extraction on ice for 30 min in a buffer containing 10 mM Tris-HCl, pH 6.8, 1 mM EDTA, 150 mM NaCl, 0.25% Nonidet P-40, 1% Triton X-100, 1 µM NaF and protease inhibitor mixture tablets (Roche Applied Science). Cell debris was removed by centrifugation at 16,000× g. The protein concentrations were determined using the BCA protein assay kit (Pierce/Thermo) and equal amounts of total protein (25–40 µg) per lane were resolved in a 4–12% gradient Bis-Tris gels (Novex, Invitrogen) under reducing conditions. After transfer to PVDF membranes (Amersham Biosciences/GE Healthcare), the filters were probed with the following antibodies: A8717 (Sigma; APP C-terminus), dNGluc (Proteintech Group Inc) and GAPDH (Millipore). After incubation with horseradish-conjugated secondary antibodies, the signal was developed using ECL Western blotting detection reagent (Pierce/Thermo).

Purified monocytes were suspended in RPMI‐1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mm L‐glutamine, 100 U mL⁻¹ penicillin, and 100 µg mL⁻¹ streptomycin (all from Sigma‐Aldrich) (henceforth referred to as “complete medium”) and seeded at three million cells per well in 6‐well polystyrene plates (Corning Life Sciences).

For FESEM analysis, the mature mono- and dual-species biofilms were established on glass coverslip in 6-well polystyrene plates (Corning, NY, USA) covered, as described above. Following biofilm formation, the harvested mono- and dual-species biofilms were washed three times with PBS and then fixed with 2.5% glutaraldehyde for 6 h at 4 °C. Subsequently, the samples were gradually dehydrated in graded ethanol (25, 50, 75, 90, 95, and 100%) for 10 min and then air-dried, gold-coated, and examined under a FESEM (Quanta FEG 650, FEI, Hillsboro, OR, USA).

The mature mono- and dual-species biofilms were established on 6-well polystyrene plates (Corning, NY, USA) as described above. After 21 h of incubation, the planktonic cells were removed with sterilized PBS. The biofilms were incubated in sterile saline solution for another 3 h at room temperature. The mixtures were subsequently sonicated and then centrifuged for 3 min at 4 °C to harvest the biofilm samples [12 (link)]. The supernatants were mixed with 1 mL of pre-cooled methanol/acetonitrile/water solution (2:2:1, v/v) successively for 1 min of vortexing, and then the mixtures were sonicated at low temperature for 30 min. Subsequently, the samples were allowed to stand at –20 °C for 60 min, followed by centrifugation at 14,000× g for 20 min at 4 °C. The supernatant was collected and vacuum freeze-dried for sample analysis. Finally, dried samples were dissolved in 100 µL acetonitrile/water (1:1, v/v), followed by vortexing and ultrasonic treatments. Next, each sample was centrifuged at 14,000× g at 4 °C for 15 min, and then each sample was transferred to a sample bottle with a lining tube for subsequent chemical analysis.

HEK293 cells were chemically transfected with pEGFP-mTRPM7 and pEGFP-mTRPM7 S1107R plasmids using polyethyleneimine (PEI) (Polysciences, Warrington, PA) or LT1 (Mirus Bio, Madison, WI) by mixing with DNA and Dulbecco’s phosphate-buffered saline (DPBS) and adding to cells in 6-well polystyrene plates (Corning Incorporated, Kennebunk, ME) (Zhelay et al., 2018 (link)). Plasmid DNA was purified using PureYield plasmid midiprep kit (Promega, Madison, WI).

This was carried out according to Oaks, Wingfield & Formal (link) (1985 (link)) using confluent HeLa cell monolayers. Briefly, HeLa cells to be used in the plaque assay were grown to 100% confluency in 6-well polystyrene plates (Corning) in DMEM supplemented with 10% FBS. One hour before the infection, cell culture medium were changed into fresh medium and Sf301 or its LPS mutants from mid-exponential phase (m.o.i 100:1) was added to the cells and subsequently incubated at 37°C for 90 min. During this adsorption or attachment phase, the plates were rocked every 30 min to assure uniform distribution of the plaque-forming bacteria. Next, an agarose overlay (5 ml) consisting of DMEM, 5%FBS, 25 µg of gentamicin per ml, and 0.5% agarose was added to each plate. The plates were incubated at 37°C in a humidified 5% CO₂ and examined daily for up to 3 days for plaque formation. The agar was carefully removed three days later and the HeLa monolayers were stained by Giemsa staining kit (Yeasen, Shanghai, China) in order to visualize the plaques.