Materials

1. RPMI 1640 1X liquid without L-glutamine, without HEPES, stored at 4°C (cat. no. 31870; Gibco Invitrogen Corporation, Paisley, UK).

2. 100 ml L-glutamine solution stored at -20°C (cat. no. G3126; Sigma, Poole, Dorset, UK).

3. 1 M HEPES solution stored at 4°C (cat. no. H0887; Sigma).

4. Nutrient Broth No. 2 (NB): 25 g dissolved in 1 L of sterile H2O and stored at 4°C (cat. no. CM67; Oxoid Ltd., Basingstoke, Hampshire, UK).

5. Oxaloacetate, pyruvate insulin (OPI) media supplement solution: one vial dissolved in 10 mL of sterile H2O stored at -20°C (cat. no. O5003; Sigma).

6. Penicillin/streptomycin solution, stored at -20°C: 10,000 IU/mL benzypenicillin sodium BP (cat. no. 301889; Britannia Pharmaceuticals, Surrey, UK) and 10,000 ^g/mL streptomycin sulphate (cat. no. 11800-038; Gibco Invitrogen Corporation, Paisley, UK).

7. Fetal bovine serum (FBS), heat-inactivated at 56°C for 30 min to destroy complement and stored at -20° C (Labtech International, Ringmer, East Sussex, UK).

8. Costar 24-well tissue culture plates (cat. no. 3524; Corning Incorporated, Ithaca, NY).

9. Costar 25-cm2, 75-cm2, and 225-cm2 vented sterile tissue culture flasks (cat. nos. 3056, 3376, 3001; Corning Incorporated).

10. Sterilin 5 mL, 10 mL, and 25 mL disposable sterile pipets (cat. nos. PP28033, PP28060, PP40125; Mackay and Lynn, Edinburgh, UK).

11. MiniPERM classic kit, molecular weight cutoff 12.5 kDa/vol 35 mL. Kit comprises: 12 production modules, single packed, sterile; four nutrient modules, reusable; four stands for the MiniPERM bioreactor (cat. no. IV-76001055; Vivascience, Sartorius AG Weender Landstrasse 94-108, 37075 Goettingen, Germany).

12. CellPROTECT, anti-shearing agent for high-density culture (cat. no. IV-76077041; Vivascience).

13. AntiFOAMa (cat. no. IV-76077320; Vivascience).

14. Universal turning device, which holds four miniPERM bioreactors (cat. no. IV-76001065; Vivascience).

15. Disposable hypodermic syringe, 50-mL Luer (cat. no. IV-76077137; Vivascience).

16. Disposable hypodermic syringe, 2-mL Luer (cat. no. IV-76077136; Vivascience).

17. Spare Luer-Lock screw caps for production modules: six pieces, single packed, sterile (cat. no. IV-76077027; Vivascience).

18. Neubauer hemocytometer (cat. no. MNK-420-010N; Fisher Scientific UK, Bishop Meadow Road, Loughborough, Leicestershire, UK).

19. Cryopreservation tubes (cat. no. 366656, Nunc A/S, DK-4000 Roskilde, Denmark).

21. Cryo 1°C freezing containers (cat. no. 5100; Nalgene, Nalge Europe Ltd, Rotherwas, Hereford, UK).

22. 2-Propanol (RH1018; Rathburn Chemicals, Walkerburn, Scotland, UK).

23. Nigrosin, dissolved to a final concentration of 0.1% w/v in phosphate-buffered saline containing 2% FBS, filter-sterilized, and stored at 4°C (BDH, Merck, Poole, Dorset, UK).

24. Supplemented RPMI culture medium: RPMI is used as the standard base medium for culturing hybridoma cell lines. The following supplements are added to each 500-mL bottle of RPMI: 10 mL of penicillin/streptomycin (see Note 1), 10 mL of glutamine (see Note 2), 5 mL of HEPES, and 50 mL of FBS (see Note 3). The contents should be mixed by swirling, not by inversion, thereby avoiding leakage and contamination. Hereafter this mixture is referred to as complete medium (CM). Two 1-mL aliquots of CM should be removed and added to two 5-mL aliquots of NB for sterility testing. Keep one NB at room temperature for 3 d and incubate the other at 37°C for 3 d. Check the NBs for evidence of microbial contamination prior to using the CM for the first time. Store CM at 4°C and use within 2 wk of preparation (see Note 2).

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