Spectrafluor plus plate reader

Human alveolar
adenocarcinoma A549
cells were maintained in DMEM, supplemented with 10% FBS and 50 U/mL
pen-strep at 37 °C with 5% CO₂. A549 cells were assayed
in Opti-MEM supplemented with 1% serum supreme and 50 U/mL pen-strep
and seeded into 96 well plates at a density of 1.5 × 10³ cells/well followed by a 6 h incubation at 37 °C, 5% CO₂. Cells were then dosed with serial dilutions of compound
and incubated for 48 h. Cell viability was determined by measuring
the conversion of resazurin to resorufin^{35 (link)} using a SpectraFluor Plus Plate Reader (Tecan). Data were fit to
an equation for a sigmoidal dose response using the equation below,
where y_i and y_f are the initial and final signal
intensities.
For comparison purposes,
the cytotoxicity
of CDDP was evaluated under the same experimental conditions. All
compounds were tested in three independent studies with quadruplicate
points. The compounds were dissolved in water, except 5, which was dissolved in DMSO and diluted in the culture medium so
that the final % DMSO was 0.4.

A549 MCTSs (diameter 600
μm) were treated by carefully replacing 50% of the medium with
drug-supplemented standard medium by using an eight-channel pipet.
In parallel, 50% of the solvent-containing medium was replaced by
solvent-free medium for the untreated MCTSs. Three MCTSs were treated
per condition and drug concentration, and the DMSO volume was less
than 1% (v/v). The MCTSs were then allowed to incubate for another
48 h. The cytotoxicity of the platinum complexes toward the MCTSs
was measured by the adenosine triphosphate (ATP) concentration with
the Cell TiterGlo kit (Promega). After 30 min of incubation, the MCTSs
were carefully transferred into black-sided, flat-bottomed 96-well
plates (Corning) and mixed with a pipet for luminescence measurements
on a SpectraFluor Plus Plate Reader (TECAN). Data were fit to an equation
for sigmoidal dose response shown above using the Prism software package.

Carbohydrates: For the extraction of starch and soluble sugars lyophilized leaf material was treated as described in (Lin et al., 1988 (link)) and determined with a coupled enzymatic assay (Stitt et al., 1989 (link)) in a Spectrafluor Plus plate reader (TECAN, Austria) in the absorbance mode.
Amino acids: Lyophilized tissue (2–8 mg) was homogenized by a TissueLyser (Quiagen) at 23 Hz for 30 s and subsequently extracted with 300 μl of 80% EtOH at 4°C for 20 min on an orbital shaker. The mixture was centrifuged at 21000 × g for 5 min at 4°C and the supernatants were transferred to fresh 2 ml test tubes, while the debris was extracted with 200 μl 96% EtOH and again incubated at 4°C for 20 min on an orbital shaker. Following centrifugation at 21000 × g for 5 min at 4°C the second supernatants were unified with the first ones in the 2 ml test tubes. The analysis of free amino acid contents was carried out as outlined in Krueger et al. (2017) (link) by reversed phase HPLC using a HyperClone^TM 5 μm ODS (C18) column (Phenomenex). The samples were precolumn-derivatized with o-phthalaldehyde according Lindroth and Mopper (1979) (link).

Cell adhesion
assay was carried
out as previously described^{47 (link)} with minor
modifications. The day before the experiment, each well was coated
with VCAM-1 (3 mg/mL) immobilized on the 96-well plate in PBS by overnight
incubation at 4 °C. Thereafter, nonspecific binding was blocked
by incubating the wells with 1% (w/v) bovine serum albumin (BSA) in
Hank’s Balanced Salt Solution (HBSS) containing 5 mM MgCl₂ at room temperature for 1 h before use. The cells were labeled
by incubation with 12.5 μM 5-chloromethylfluorescein diacetate
(CMFDA) in HBSS without 1% BSA at 37 °C for 30 min. The labeled
cells were then centrifuged at 1000 rpm and washed twice with HBSS
containing 1% BSA to remove excess CMFDA. Labeled cells (about 1 ×
10⁵ cells/well) were plated on each well in the presence
or absence of visabrons and incubated at 37 °C for 60 min. Unbound
cells were removed by washing the wells three times with 1% (w/v)
BSA in HBSS, and bound cells were lysed by the addition of 0.5% Triton
X-100 (diluted in DDW). The fluorescence in each well was quantified
with a SPECTRAFluor Plus plate reader (Tecan), at λ_ex = 485 nm and λ_em = 530 nm. To determine the number
of adhered cells from the fluorescence values, a standard curve was
generated by serial dilution of known numbers of CMFDA-labeled cells.