A SAC that detects such minor defects in kinetochore microtubule occupancy caused by an imbalance of microtubule stability within the spindle would be able to indirectly probe for plate positioning, giving cells time to correct this imbalance and ensure a symmetric metaphase plate position.
The probe and plate were disinfected by means of a plasma sterilization procedure between uses.
This second (probe-optimization) plate is run in the GeneAmp 5700 sequence detection system using the same thermocycler program as used for the first (primer-optimization) test plate.
As suggested by the reviewers, we pursued the more sensitive technique of using I-labeled CTB to probe TLC plates in an attempt to detect GM1 in Colo205 and T84 cells.
In these experiments, the local temperature was measured using fibre-optic probes placed adjacent to the plate (probe TS 5; evaluation unit FoTemp 1; OPTOcon, Dresden, Germany).
On day 10, animals were bled and the sera of individual animals examined for HA-specific IgG by probing ELISA plates coated with the PR8 virus (Figure 5A).
Probe-optimization plates were also run in the GeneAmp® 5700 sequence detection system using the same thermocycler program as used for the primer-optimization plates.
After 18 hours, another batch of 3T3 cells, which were also seeded with fluorescent probes, were plated on top of the confluent cell layer.
Then, we added CNQX (10 μM, 6-cyano-7nitroquinoxaline-2,3-dione, Sigma), AMPA hydrobromide (40 μM, α-Amino-3-hudroxy-5-methylisoxazole-4-propionic acid, Sigma) or Glutamate (100 μM, Sigma) in a calcium buffer solution containing 5 mM CaEGTA, 5 mM EGTA, 100 mM KCl and 30 mM MOPS with free 0.15 μM Ca2+ (Molecular Probes) and plates were measured immediately.
