SP2/0-Ag14 (AC-free)

Fusion partner

Mouse x mouse hybridoma non-secreting, serum-free, animal component (AC) free

Sp2/0-Ag14 cell line (Sigma Catalogue number. 85072401) adapted to grow in animal component free medium (EX-CELL Sp2/0 Serum-Free Medium for Sp2/0 Cells Chemically Defined, Sigma cat no 14660C). Sp2/0-Ag14 is a non-Ig-secreting or synthesising line derived from a cell line created by fusing a BALB/c mouse spleen cell and the mouse myeloma P3X63Ag8. Resistant to 8-azaguanine at 20ug/ml and does not survive in HAT containing media. Can be used as a fusion partner for generating hybridomas.

EX-CELL Sp2/0 Serum-Free Medium (Sigma cat no. 14660C) + 8mM Glutamine. When freezing cells down use 50:50 fresh culture medium:conditioned medium plus 10% DMSO)

Viability may be poor on resuscitation and may initially decrease further. Full recovery may take up to 2 weeks. A centrifugation step to remove the cryoprotectant is essential. Rapidly thaw the frozen ampoule in a water bath at 37°C for 1-2 minutes. Transfer the contents to a centrifuge tube and slowly add 5-10ml of pre-warmed growth media. Remove a sample for counting. Centrifuge at 100g for 2-3 minutes to pellet cells and seed at a relatively high density of 5-7 x 10⁵ cells/ml. Leave culture flask upright and observe regularly until viable proliferating cells are seen. Once established use a split ratio of 1:2 approximately every 4 to 5 days; 5% CO₂; 37°C..

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