After 24 h, cells were incubated with mouse anti-FLAG-M2-Cy3 antibody (Sigma-Aldrich Co). at 4°C and stimulated with 20 nM CCL2 at 37°C for the times indicated before being fixed, and incubated with goat anti-FLNa (Santa Cruz Biotechnology, Inc).
The cells are forced to float in suspension for the times indicated before being harvested for analysis.
Mixed cortical cultures were deprived of glucose or incubated with glucose for the times indicated before being washed and exposed to either 25 µM NMDA or 100 µM glutamate in HCSS at room temperature.
If you didn't make it back before the time indicated on the pass, you could be fined or imprisoned.
4. Meanwhile, cook the noodles uncovered in the boiling water until al dente according to the instructions on the pasta packaging (check the pasta 1 to 2 minutes before the time indicated to make sure it isn't already done).
Subsequent CD4 determinations performed before the time indicated by the rule (tnext) were counted, compared with the threshold, and discarded as superfluous.
Cells were stimulated either with 1 mg/ml OKT3 or 6000 units/ml Roferon-α and incubated at 37 °C for the amount of time indicated before being transferred on to ice and lysed immediately with 1 ml of ice-cold lysis buffer as above.
Cook al dente, using the timing instructions on the package as a guide but checking the pasta a minute before the time indicated is up.
To measure the TG synthesis and secretion rate at the times indicated in the figures, four hours before terminating the experiment, [1,2,3-H]glycerol (13 pCi/mL) was added to the apical chamber.
Caco-2 (a) HCT-8 (b) and HT-29 cells (c) were incubated with DiO-labelled OMVs from strains LB226692 or C227-11Φcu for 24 h and fluorescence was measured at the times indicated using FACScan flow cytometer before (total cell-associated OMVs) and after trypan blue (TB) quenching (internalised OMVs).
Agonists were then added for the times indicated (0.1 nM–3 µM, triplicate wells) before incubations were terminated by fixation with 3% paraformaldehyde in phosphate-buffered saline (PBS, 10 min at 21°C).
