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Bioprotocols
Drug Discovery Protocols: This database contains a comprehensive list of various bioprotocols that are used in biomarker discovery. These complete protocols include techniques used in molecular and protein drug discovery research. We have listed unique protocols that are not available in any other online bioprotocol database.
Cell culture and cell isolation protocols

Preparation of a Mouse Spleen Cell Suspension and the Activation and Enrichment of T Cells





Mouse spleen was immersed in 8 ml of Hank's balanced salt solution (HBSS), gently minced with a sterile cover slip, transferred to a 15 ml centrifuge tube (Costar), and spun at 200timesg for 5 minutes. The supernatant was discarded and the cell pellet was resuspended in the residual buffer by gently tapping the wall. The contaminating red blood cells (RBC) were lysed by the addition of 1 ml of RBC lysis buffer (0.6 M NH 4Cl, 0.17 M Tris-base, pH 7.65), followed by a 2 min incubation at room temperature and rapid quenching with 9 ml of HBSS. The cells were pelleted at 200timesg for 5 minutes, washed twice and resuspended in RPMI medium. The concentration and viability of cells in the mixture were determined with a hemocytometer (Cambridge Scientific Inc.) and Trypan blue exclusion.

The spleen cells were adjusted to a final concentration of 3times10 6/ml with RPMI medium and Concanovalin A was added to a final concentration of 2 micrograms/ml to activate the T cells. The cell suspension was transferred to a 6-well culture plate (5 ml/well) or a 10-cm culture dish (10 ml/dish) and incubated at 37 degree C., 5% CO 2 for 48 hours before harvesting. The activated spleen cells, including activated T cells, were resuspended in 5 ml of HBSS and carefully overlaid on top of a 5 ml 55% cushion of Percoll solution in a centrifuge tube. Care was taken not to disturb the separated layers. The cells were spun at 1,900timesg for 13 minutes at 25 degree C. without the brake. The enriched T cells were collected from the interface of the two layers, washed twice with HBSS, and were ready for experiments.

Source: 7,744,888



Disclaimer: Sciclips does not guarantee the authenticity or validity of the protocols listed in this section. These protocols are extracted "as is" from various sources, with minor modifications. Please refer to the respective citations, which are hyperlinked, for more details. The use of cited molecules, not limited to proteins, nucleic acids, compounds etc, in the protocols that are extracted from patents may be covered under patent rights. These molecules should be used for information purpose only.
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