Isolation of cell nuclei for the application in the cell-free system
PREPARATION OF ISOLATED NUCLEI - PROCEDURE 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 礐i/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.
MATERIAL 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 礸/ml Leupeptin
2 礸/ml Pepstatin
2 礸/ml Aprotinin10 ml of 50 mM PIPES, pH 7.4
500 祃 of 1 M KCl
1.0 ml of 100 mM MgCl
50 祃 of 1 M DTT
500 祃 of 10 mM PMSF
100 祃 of 1 mg/ml Leupeptin
100 祃 of 1 mg/ml Pepstatin
100 祃 of 1 mg/ml Aprotinin
37.65 ml H2O nuclease free10 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 礸/ml Leupeptin
2 礸/ml Pepstatin
2 礸/ml AprotininGrow 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 祃 of 4.2 mM Cytochalasin B (CB); Incubate 30 min at 37癈 (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 礛 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 祃 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 祃 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 祃 aliqots (50 000 nuclei).
Store nuclei at -70癈 in aliquots until required (up to several weeks).
