Coulter multisizer 3

AuMBs were produced using the following emulsion method: a sonicator (Model 102C, Branson, Danbury, CT, USA) was used to sonicate a premixed solution—which contained 4% (w/v) HSA (human serum albumin, Octapharma, Vienna, Austria), 6 nM AuNPs, phosphate-buffered saline (PBS), and C₃F₈ gas—for 3 min followed by resting on ice for 5 min. Centrifugation was then performed at 1100 rpm for 3 min. The concentration and size distribution of the AuMBs were measured by a cell counter/analyzer using a 30-µm aperture (Coulter MultiSizer III, Beckman Coulter, Brea, CA, USA). AuMBs with a mean size of 1–3 µm were selected by varying the centrifugation parameters.

To assess proliferation, cells were seeded at 4.2 × 10⁵ cells/mL in 24-well plates. Following 24 h, the media were replaced with fresh RPMI + 5% FCS, in the presence or absence of treatments and a selection of wells counted (day 0 counts). Cells were subsequently counted each day up to 7 days to assess proliferation. Briefly, this entailed the removal of old media and the addition of trypsin/EDTA in order to lift cells before passing gently through a 25 G needle to achieve a single-cell suspension. The resulting solution was then added to isoton in a counting cup and the cell number was determined using a Coulter Multisizer III (Beckman Coulter Life Sciences, Indianopolis, IN, USA) Each well was counted twice with all conditions performed in triplicate.

Perfluorobutane gas-filled lipid MBs carrying either Silencer Select siRNA (Ambion) against luciferase or a negative control sequence were prepared as previously described [16 (link), 17 (link)]. Briefly, chloroform solutions of 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-ethylphosphocholine, 1,2-distearoyl-sn-glycero-3-phosphoglycerol (Avanti Lipids), and polyethylene glycol-40 stearate (Sigma) at a mole ratio 100:43:1:4.5 were dried with argon gas. An aqueous micellar solution of lipid was then prepared by adding phosphate buffered saline (PBS) containing 1 mmol/L EDTA and the solution was sonicated (Misonix) to disperse lipids. The resulting lipid solution was diluted 1:4 in PBS/EDTA, and 8 μg of Silencer Select siRNA against luciferase or a negative control sequence was added. The solution was placed in a sealed glass vial and the head space was filled with perfluorobutane gas (FlouroMed), followed by amalgamation (Bristol Myers-Squibb) to form perfluorobutane gas-filled MBs [17 (link)]. The resulting siRNA-loaded MB solution contained approximately 7×10⁸ MB/mL with a mean diameter of 2.1±1.1 μm as measured by a Coulter Multisizer 3 (Beckman Coulter).

Subcutaneous and visceral adipose tissue samples were fixed in 2% osmium tetroxide and shaken at 37°C for 7 days. The fixed tissues were filtered through a 250-μm and 25-μm mesh to obtain a single cell suspension. The diameter and distribution of adipocytes were measured using a Coulter Multisizer 3 particle counter (Beckman Coulter, Inc., Brea, CA, USA).

For each taxon, we averaged the number of stamens and ovules per ovary after dissecting one floral bud from 5 to 21 individual plants under a dissecting microscope totaling 59 flower buds (Table 3). For each sample, we also estimated the number of pollen grains per anther by squashing one anther randomly selected on a slide containing Isoton II (Beckman Coulter, Fullerton, CA) and counting the number of pollen grains contained in aliquots of 500 μl using a particle counter (Coulter Multisizer 3, Beckman Coulter). Pollen production per flower was estimated as the number of pollen grains per anther (mean value of three aliquots) multiplied by the number of anthers in the flower; then, we computed the pollen‐to‐ovule (P/O) ratios accordingly (Cruden, 1977). We also dissected 1–3 recently‐opened flowers from 7 to 10 individual plants per taxon to measure: (a) length of petals, (b) height of the tallest anther, (c) height of the ovary, and (d) height of the stigma; then, we computed the stigma‐anther separation (i.e., herkogamy) by subtracting the height of the tallest anther from the height of the stigma (ovary size plus style length). Stigma‐anther separation has positive values when stamens do not reach the stigma height (i.e., approach herkogamy) and negative values when the stigma is below the anthers (i.e., reverse herkogamy).

Targestar^®-P (Targeson; San Diego, CA) was used in all studies. Targestar-P (TS-P) is a lipid encapsulated decafluorobutane (C₄F₁₀) microbubble (MB), and is commercially available for use as an imaging agent in life science research. Targestar-P is a polydisperse formulation, containing MB of diameter between 1 and 8 um. An “up-sized” preparation of Targestar-P was prepared by differential centrifugation, as in Shekhar et al (18). Following centrifugation, MB were packaged in depyrogenated glass vials under a headspace of decafluorobutane and stored at 4-8°C. MB size distribution and concentration was assessed by electrozone sensing, using a Coulter Multisizer 3 (Beckman Coulter, Fullerton, CA, USA), which measures MB in the range 0.6–18 μm.