Sucrose

A previously described protocol to freeze glioblastoma spheroids [38 (link)] was modified. Briefly, at the desired time point, collagen gels were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS (v/v) overnight at 4°C. The collagen gels were gently detached from glass squared cells and submerged in 7 ml of 30% sucrose (Sigma) in 10X PBS and stored at 4°C for approximately 24 h. The gels were then submerged in a mixture of 30% sucrose and OCT (8:2 sucrose to OCT for 2 mg/ml and 7:3 ratio for 6 mg/ml) and frozen down on a dry ice/ethanol bath.

For acclimation to the sucrose solution, 1% sucrose (Sigma, St. Louis, MO, USA) solution and tap water contained in two bottles were provided to the individual rats for 24 h. The volume of both the sucrose solution and tap water was the same (250 mL), and the remainder of both the sucrose solution and tap water were checked to measure consumption. The initial positions of the two bottles were randomized to prevent a positional preference, and after 24 h, the positions of the bottles were switched to avoid adjustment to the position of the liquids.

Pea aphids (Acrythosiphon pisum strain LSR1) were grown as an all-female clone on Fava bean (Vicia faba) seedlings on 16h/8h light/dark cycles at 20°C. Once reaching adulthood, apterous adults were raised on Fava bean plants on 16h/8h light cycles and allowed to reproduce overnight. After seven days, all aphids, in the 4^th larval instar and typically amounting to 5g, were removed from the Fava bean plants. Aphids were weighed and surface-sterilized in 0.5% NaClO solution, then rinsed twice in Ultrapure water (MilliporeSigma), each 30 s. Aphids were gently ground in a mortar and pestle in 40mL sterile Buffer A (25mM KCl (Sigma-Aldrich), 35mM Tris base (Sigma-Aldrich), 10mM MgCl₂ (Sigma-Aldrich), 250mM anhydrous EDTA (Sigma-Aldrich), and 500mM Sucrose (Sigma-Aldrich) at pH 7.5). Aphid homogenate was vacuum filtered to 100μm, then centrifuged at 1500g for 10 min at 4°C. Supernatant was discarded, and the resulting pellet was resuspended in 20mL Buffer A and vacuum-filtered three times from 20μm, to 10μm, and finally to 5μm. The resulting filtrate was spun at 1500g for 30 min at 4°C and supernatant discarded. The resulting pellet was resuspended in 10mL Sucrose solution (300mM Sucrose (Sigma-Aldrich) and 100mM Tris base (Sigma-Aldrich)) then checked on a brightfield microscope for intact Buchnera cells. Buchnera cells remain intact while at 4°C for a maximum of 24h.

60% (w/w) sucrose (Sigma-Aldrich, cat. no. S0389-1KG) in 1× PBS, pH 7.4 (Thermo Fisher, cat. no. 10010023) (filter sterilized) RI: 1.442 nD20
25% (w/w) sucrose (Sigma-Aldrich, cat. no. S0389-1KG) in 1× PBS, pH 7.4 (Thermo Fisher, cat. no. 10010023) (filter sterilized) RI: 1.372 nD20
15% (w/w) sucrose (Sigma-Aldrich, cat. no. S0389-1KG) in 1× PBS, pH 7.4 (Thermo Fisher, cat. no. 10010023) (filter sterilized) RI: 1.356 nD20

1-Palmitoyl-2-oleoylglycero-3-phosphocholine
(POPC), egg sphingomyelin (SM), cholesterol (Ch), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)(ammonium salt) (Rho-DOPE) were
purchased from Avanti Polar Lipids (Alabaster, AL). Poly(1,2-butadiene)-b-poly(ethylene oxide) (PBD₂₂-b-PEO₁₄) with the average molecular weights of 1200 and
600 g/mol for the PBD and PEO blocks, respectively, was purchased
from Polymer Source Inc. (Dorval, Quebec, Canada). Sucrose, glucose,
and chloroform were purchased from Sigma-Aldrich (St. Louis, MO). N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium
salt (NBD-PE) and 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose
(2-NBDG) were purchased from ThermoFisher Scientific (Eugene, OR).
Sucrose, glucose, and 2-NBDG solutions were prepared in deionized
water (DI) (Millipore, Sigma, St. Louis, MO) were used for experimentation.