L7 55

Ls 2362 was grown in MBS broth^{25 (link)}, and Bti strains 4Q5, 4Q7/pWF45, 4Q7/pBU-cyt1Aa-binA, and 4Q7/p45S1 were grown in 50 ml of NBG^{13 (link), 14 (link)} appropriately supplemented with 25 μg/ml erythromycin and 3 μg/ml tetracycline, at 28 °C for 4 days by which time >95% of the cells had sporulated and lysed. Spores and crystals were collected by centrifugation at 6,500 g for 15 min, washed 2x in double-distilled (dd) H₂O, followed by centrifugation at 6,500 g for 15 min at 4 °C after each wash, and lyophilized (FreezeZone 4.5, Labconco) for storage.
To isolate parasporal bodies, spore/parasporal body mixtures collected from 50 ml cultures were resuspended in 15 ml ddH₂O and sonicated twice at 50% duty cycle for 15 s using the Ultrasonic Homogenizer 4710 (Cole-Parmer Instrument Co.). Five-milliliter samples were loaded onto a sucrose gradient cushion (30–65% w/v), which was then centrifuged at 20,000 g for 45 min at 20 °C in a Beckman L7–55 ultracentrifuge using the SW28 rotor. Bands containing parasporal bodies were collected and washed twice in ddH₂O, followed by centrifugation at 6,500 g for 15 min at 4 °C after each wash and lyophilized for storage.

Three mL of high-titer phage preparation were centrifuged using the preformed CsCl gradient (1.25 g/mL, 1.4 g/mL, 1.5 g/mL and 1.7 g/mL, 2.5 mL each) at 10 °C, 25,000 rpm, for 2.5 h in a Beckman Coulter ultracentrifuge, L7-55, using a SW 41 Ti rotor. Further, ten µL of the concentrated phage suspension (10⁹ PFU/mL) were applied to carbon-coated copper grids (400 mesh) and negatively stained with 1% uranyl acetate. The grids were analyzed using a JEM 1200EX (JEOL, Tokyo Japan) transmission electron microscope at 80 kV accelerating voltage. Images were taken on Kodak film SO-163 (Kodak, Cat. \# 74144, Hatfield, PA, USA). Phage particle dimensions were measured using ImageJ version 1.53e in relation to the scale bar generated by the microscope.

Lentiviral vectors mCherry-hCdt1(30/120)/pCSII-EF-MCS (DDBJ/EMBL/GenBank, AB512478) and mVenus-hGeminin(1/100)/pCSII-EF-MCS (DDBJ/EMBL/GenBank, AB512479) were purchased from the Riken Brain Science Institute, Japan (Dr. Atsushi Miyawaki, head of provider laboratory, and Dr. Hiroyuki Miyoshi, developer of pCSII-EF-MCS). Lentiviral particles were generated by co-transfection of HEK-293TN cells (System Biosciences) with mCherry-hCdt1 (30/120)/pCSII-EF-MCS or mVenus-hGeminin (1/100)/pCSII-EF-MCS lentiviral vectors, alongside the packaging plasmid psPAX2 (Addgene plasmid, #12260) and the envelope plasmid pMD2.G (Addgene plasmid, #12259). The culture supernatant was collected and concentrated by ultracentrifugation at 22,000 rpm for 3 h (Beckman L7-55 with SW32Ti rotor) at 4 °C. The virus titer was estimated by transduction on HeLa cells and subsequent flow cytometric analysis for fluorescent protein expression.

Whole bodies of larvae were finely chopped and mixed to obtain as much homogenous material as possible. After that, 0.2 g was taken to determine the concentration of thiobarbituric acid reactive substance (TBARS) as a standard marker for LPO (lipid peroxidation); the rest was used for determination of antioxidant parameters. Tadpoles were individually homogenized with an Ultra Turrax homogenizer T-18 (IKA-Werk, Staufen, Germany) at a 1:5 ratio in an ice cold 25 mM sucrose buffer (pH 7.4) containing 10 mM Tris-HCl and 5 mM EDTA. The homogenates were sonicated with an ultrasonic homogenizer (Sonopuls HD 2070, Bandelin electronic, Berlin, Germany) for 30 s at 10 kHz. A part of the sonicate was taken for measurement of total reduced glutathione (GSH) concentration, while the rest was centrifuged in an L7-55 ultracentrifuge (Beckman, Brea, CA, USA) at 100,000× g at 4 °C for 90 min [32 (link)]. The supernatants were used for measuring AOS parameters.

For transmission electron microscopy (TEM) analysis, the high-titer phage preparation underwent CsCl density gradient centrifugation. In this procedure, two mL of the phage preparation was meticulously layered onto a preformed CsCl gradient with specific densities of 1.3 g/mL, 1.4 g/mL, 1.5 g/mL, 1.6 g/mL, and 1.7 g/mL, each occupying 2 mL of the gradient. Ultracentrifugation was carried out using a Beckman Coulter L7-55 ultracentrifuge equipped with an SW 40 Ti rotor. The centrifugation process was conducted at a temperature of 18 °C, with a centrifugal force of 110,961.5 g for a duration of 1.5 h.
Following the density gradient centrifugation, five microliters of the phage suspension was loaded onto 400 mesh carbon-formvar-coated copper grids and negatively stained with 1% uranyl acetate. Phage particles were visualized using a JEM-100C transmission electron microscope (JEOL, Akishima, Japan) with an accelerating voltage of 80 kV. Images were taken on Kodak film SO-163 (Kodak, Cat. #74144, Hatfield, PA, USA) with 45,000× magnification. Phage particle dimensions were measured for 10 phage particles using ImageJ version 1.53e [56 (link)] in relation to the scale bar generated from the microscope.

Thoroughly washed plants were finely cut or chopped using a mechanical blender, and soaked in an equal quantity of M63 medium supplemented with 0.4% DIECA. Plant debris was removed by filtration and subsequent centrifugation at 85,000× g for 0.5 h at 4 °C in a Beckman ultracentrifuge (L7-55, rotor SW28). The extract was then sterilised by filtration through a 0.22 μm nitrocellulose filter (Millipore, Burlington, MA, USA) and stored at −80 °C for further use.