Cell Suspension Production

1. Two days before the actual separation procedure, transfer the carcinoma cells to new flasks to keep them in log phase growth (see Note 1). Use Accutase as described in step 2.

2. On the day of the separation, check that the carcinoma cells are subconfluent in the flasks and then detach cells from the flask using Accutase diluted in cold PBS. Follow the manufacturer's instructions for optimum concentration and incubation time (see Note 2).

3. Wash cells twice with labeling buffer by centrifugating at 300g and proceed to determine cell concentration using a particle counter. Also, perform a viability test using a Trypan blue exclusion method. Keep cells in cold storage (4°C) until used.

4. Buffy coat (white blood cell fraction) obtained from Red Cross must be kept in cold storage until use. Take an aliquot of the buffy coat and dilute with HBSS to determine cell concentration. Either a hemacytometer or an automated particle counter can be used for cell count (see Note 3).

5. Based on the total number of cells present in the buffy coat, seed viable carcinoma MCF-7 cells to a final percentage of 0.1% (see Notes 4 and 5).

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