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Buffers and solutions(All solutions are supplemented with Complete Protease inhibitor tablet (Roche, Cat no: 1-873-580) at the final concentration of 1 tablet/50ml):PBSBuffer A:10 mM Hepes, pH 7.9, 10mM KCl, 1.5mM MgCl2, 0.5mM DTTS1 solution: 0.25 M Sucrose, 10 mM MgCl2S2 solution:0.35 M Sucrose, 0.5 mM MgCl2S3 solution:0.88 M Sucrose, 0.5 mM MgCl2See \"Notes” below on making stock sucrose solution.

Procedure

Note: Nucleoli were prepared from HeLa cells using a variation on a method described by Muramatsu and co-workers in 1963(Muramatsu M, Smetana, K., and Busch, H.: Quantitative aspects of isolation of nucleoli of the Walker carcinosarcoma and liver of the rat. Cancer Res. 1963; 25:693-697).1. Seed HeLa cells (ATCC number: CCL-2) on to 10x14 cm Petri dishes and culture at 37oC in 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) containing 4mM L-glutamate, 4.5 mg/ml glucose and 0.11 mg/ml sodium pyruvate (Invitrogen UK, Cat no: 41966-029), supplemented with 100 U/ml Penicilin and 100 µg/ml Streptomycin (1% v/v Penicilin/Streptomycin solution, Invitrogen UK, Cat no: 15140-122) until 90% confluence (approx. 107 cells per dish). This number of HeLa cells consistently provides nucleoli with excellent yield and purity. It is possible to scale down the preparation, although purity of the isolated nucleoli may suffer. Make sure you monitor every step using a phase contrast microscope (see below). 1 hour before nucleolar isolation, replace with fresh, pre-warmed medium.2. Harvest cells by trpysinization. Rinse each dish 3X with pre-warmed PBS, and on removal of the last rinse, add 2 ml of trypsin-EDTA solution (Invitrogen UK, Cat no: 25300-054) per dish. Swirl the dishes to make sure the trypsin-EDTA is evenly distributed, and return the dishes to the incubator for about 5 min. Check under a phase contrast microscope that all the cells are detached. Prolong incubation if needed. Add into each dish 8 ml of pre-warmed medium, pipette up and down so that all the cells are collected as a single-cell suspension. Pool all the harvested cells into 2x 50ml Falcon tubes. For some strains of HeLa cells, it is also possible to harvest the cells by scraping them in 5 ml ice-cooled PBS per dish. Since scraping may lead to impure nucleolar preparation in some HeLa strains, it is not recommended as the method of first choice.3. Wash 3 X with ice-cold PBS at 218 g (1000 rpm, Beckman GS-6 centrifuge, GH-3.8 rotor) at 4oC.4. After the final PBS wash, resuspend the cells in 5ml of Buffer A and incubate the cells on ice for 5 min. Put a small drop of the cell suspension on a glass slide and check under a phase contrast microscope, such as a Zeiss Axiovert 25, using a 20X objective. The cells should be swollen, but not burst (Fig 1). Nucleoli of cultured mammalian cells disassemble at 37oC in hypotonic conditions (Zatsepina et al, 1997). It is therefore imperative to keep the cell suspension on ice during this step.

Figure 1: HeLa cells after step 4. Note the swollen cytoplasm and prominent nucleoli. Bar: 10µm.

5. Transfer the cell suspension to a pre-cooled 7 ml Dounce tissue homogenizer (Wheaton Scientific Product Cat no: 357542). Homogenize 10 times using a tight pestle (\"A” specification: 0.0010 - 0.0030 clearance), while keeping the homogenizer on ice. The number of strokes needed depends on the cell type used (see \"Notes”). It is therefore necessary to check the homogenized cells under a phase contrast microscope after every 10 strokes. Stop when 90% of the cells are burst, leaving intact nuclei, with various amounts of cytoplasmic material attached. In most cases, the presence of this cytoplasmic contamination does not affect the final purity of the isolated nucleoli (Figure 2).

6. Centrifuge the homogenized cells at 218g (1000 rpm, Beckman GS-6 centrifuge, GH-3.8 rotor) for 5 min at 4°C. The pellet contains enriched, but not highly pure, nuclei.