Protocols

Isolation of cell nuclei for the application in the cell-free system

Posted on Monday, November 24, 2003

Description
Pretreatment of epithelial cells with 20 µM Cytochalasin B for 30 min in RPMI prior to harvesting results in the reduction of cytoskeletal microfilaments and the easier release of the nuclei from the surrounding cytoskeleton during cell lysis. For the preparation of Jurkat nuclei, the step of cytochalasin treatment can be omitted. Input of about 1E+07 cells. Lysis in hypotonic medium (about 23 mM compared with 137 mM in isotonic medium) using a Dounce homogenizer (B type pestle: clearance about 0.7 mm) and purification of free nuclei by centrifugation through sucrose solution of medium concentration (30% = 0.88 M). Yield: between 30% and 50% is a good yield.

Procedure
PREPARATION OF ISOLATED NUCLEI - PROCEDURE

Grow cells in a 160 cm2 flask (25 ml medium) up to about 75% cell density.
Remove medium from the cell culture flask except 5 ml. To the remaining 5 ml add 25 µl of 4.2 mM Cytochalasin B (CB); Incubate 30 min at 37°C (cells will show an alterered morphology afterwards).
Remove the CB containing supernatant and harvest cells by trypsinization and centrifugation at 200 g for 10 min.
Wash cells two times with 10 ml PBS, pH 7.2.
Wash cells once with 5 ml Nuclei Buffer (NB).
Resuspend cells in 10 volumes of NB (including 10 µM Cytochalasin B) (total volume should be at least 1,5 ml if you use a 2 ml Dounce homogenizer).
Let cells swell on ice for 20 min to 30 min (control swelling process under microscope).
Gentle lysis with a Dounce homogenizer:
About 25 to 50 controlled, but determined strokes, depending on cell type; check liberation of nuclei and grade of purity of nuclei by examination of sample under phase contrast microscope.
Layer liberated nuclei over 30% sucrose (0.88 M) in NB:
About 500 µl lysate over 2 ml 30% sucrose in a 6 ml round bottomed PP centrifuge tube.
Spin nuclei down at 800 g for 10 min; aspirate supernatant, then suck away the top layer and the interphase, thoroughly.
Optional for higher purity of nuclei:
take nuclear pellet up in 500 µl NB and layer nuclei a second time over 30% sucrose (0.88 M) in NB. Spin down at 800 g for 10 min, and aspirate supernatant.
For a wash step, take nuclear pellet up in 3 ml NB (at this point take a sample for counting nuclei in hemcytometer under phase contrast microscope), and centrifuge at 800 g for 10 min, aspirate supernatant.
Resuspend nuclei pellet in nuclei storage buffer (NSB) at 2E+08 nuclei/ml or, in case of radioactive labelled nuclei: resuspend in NSB at 1E+07 nuclei/ml and distribute the suspension in 5 µl aliqots (50 000 nuclei).
Store nuclei at -70°C in aliquots until required (up to several weeks).




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Preparation of radioactive labeled nuclei

For the preparation of radioactive nuclei, ALVA31 cells were harvested and 5x106 cells were reseeded in 162 cm2 tissue culture flask in complete RPMI + 2 µCi/ml [3H]-thymidine. After 24 h of incubation, labeled cells were harvested and the nuclei were prepared from those cells as described in the protocol above. The degree of labeling was about 1 cpm/nucleus.



Recipes
NUCLEI BUFFER (NB):
COMPOSITION: RECIPE for 50 ml:
10 mM PIPES (pH 7.4)
10 mM KCl
2 mM MgCl2
1 mM DTT
0.1 mM PMSF
2 µg/ml Leupeptin
2 µg/ml Pepstatin
2 µg/ml Aprotinin
10 ml of 50 mM PIPES, pH 7.4
500 µl of 1 M KCl
1.0 ml of 100 mM MgCl
50 µl of 1 M DTT
500 µl of 10 mM PMSF
100 µl of 1 mg/ml Leupeptin
100 µl of 1 mg/ml Pepstatin
100 µl of 1 mg/ml Aprotinin
37.65 ml H2O nuclease free
NUCLEI STORAGE BUFFER (NSB):
COMPOSITION: RECIPE for 1 ml:
10 mM PIPES (pH 7.4)
5 mM EGTA
80 mM KCl
20 mM NaCl
50% Glycerol
250 mM Sucrose
1 mM DTT
0.2 mM Spermine
0.5 mM Spermidine
0.1 mM PMSF
2 µg/ml Leupeptin
2 µg/ml Pepstatin
2 µg/ml Aprotinin
200 µl of 50 mM PIPES (pH 7.4), incl.
25 mM EGTA
80 µl of 1M KCl
20 µl of 1M NaCl
500 µl Glycerol
85 mg Sucrose
1 µl of 1M DTT
10 µl of 20 mM Spermine
10 µl of 50 mM Spermidine
10 µl of 10 mM PMSF
2 µl of 1 mg/ml Leupeptin
2 µl of 1 mg/ml Pepstatin
2 µl of 1 mg/ml Aprotinin
106 µl H2O nuclease free


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