P-FPMI-56 - P-FPMI-56

Time Course and GpC Treatment For Resting Monocytes

1. Wash newly isolated monocytes once in 15 ml PBSA (ie. No EDTA). Centrifuge the 15 ml centrifuge tube for 10 minutes at 1000 rpm at 10 oC.

Resuspend monocytes 107 per ml in culture medium (Aim V + 2% FBS heat inactivated + 50 µM 2Me, + 2 mM L-glutamine). Add 0.5 ml of cells (5 million CD14+) to each well on a 12 well Costar Tissue Culture plate. Add 2.5 ml of culture medium to each well to make the final volume 3 ml.

2. Rest the cells for 20h 37oC with 5% CO2 and 96% humidity in air.

3. Add 2 micrograms/ml GpC ODN on to the media.

4. Incubate the plate(s) for 4h at 37oC with 5% CO2 and 96% humidity in air.

5. Following each incubation period remove culture supernatant from each well into a clean 15 ml Corning centrifuge tube. Wash cell monolayer 2x with 1 ml PBSA (no EDTA). Transfer the wash solution from each well into its own 15 ml centrifuge tube. Add 500 µl of Trizol (Sigma) to each well prior to centrifugation of the wash supernatant. Centrifuge the tubes containing wash solution for 1400 rpm for 5 minutes at 4 oC. Discard supernatant and resuspend cells in 100 µl of Trizol (Sigma). Add the Trizol resuspension to the corresponding well from which the cells originated. Seal plate with parafilm and freeze at -20 oC.

All experiments using protocol P-FPMI-56: (E-BUGS-112, E-FPMI-2)