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In parallel experiments, tubes were incubated with FITC-conjugated anti-CD107a (BD Biosciences) at the beginning of the incubation, to determine degranulation as a consequence of stimulation.
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Starting with Tube 0 (the storage aliquot) with 1,000 μL, 900 μL was transferred to Tube 1; 800 μL was then transferred from Tube 1 to Tube 2 and so on until 200 μL was transferred from Tube 7 to an eighth tube not used in the study; thus, the tubes used in the experiment (tubes 0 to 7) each contained 100 μL of pooled CSF.
In sterol depletion experiments, pollen tubes were incubated with 16 mM BCD for 2 h before loading the dye.
For the knockdown experiments, pollen tubes were first fixed with 4% paraformaldehyde, 50 mM PIPES and 10% sucrose, vacuum infiltrated for 5 min and incubated for 1 h.
For this experiment, four tubes filled with pure India ink were placed at different depths with 1 cm of distance between each other.
Experiments of tube expansion in a square die using a self-designed apparatus are also conducted.
Although many publications have discussed method validations, few detail how to perform experiments for tube verification and validation.
And it turns out that, in order to bring particles up to the relativistic velocities required for certain experiments, a tube longer than the circumference of the Earth would be needed.
In our experiments, the tube was flushed for at least 20 min before treatment with N2, followed by 15-min flushing with 1 1 acetylene/N2.
Before each experiment, the tubes of the system were cleaned, washed with 70% EtOH and then rinsed with PBS and RM.
In each experiment, duplicate tubes were set up for control as well as each dilution of the decoction.
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