Experiments on the preparation of nuclei from liver tissue
Experiments on the preparation of nuclei from liver tissue
This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.
Operation method
Preparation of nuclei from liver tissue
Materials and Instruments
Rats Move 1. Prepare lysis buffer and concentrated sucrose solution (PMSF added prior to use) and place on ice. For more product details, please visit Aladdin Scientific website.
Lysis buffer Concentrated sucrose solution
Ultracentrifuge Tissue grinder
Lysis buffer:
0.25 mol/L sucrose reticulocyte standard buffer (RSB)
0.25 mol/L sucrose (super pure)
10 mmol/ Tris-HCl (pH 7.4)
10 mmol/L NaCl
3 mmol/L MgCl2
1 mmol/L DTT
0.5 mmol/L PMSF (added before use from 100 mmol/L storage solution dissolved in ethanol)
Concentrated sucrose solution:
2 mol/L sucrose RSB
2 mol/L sucrose (super pure)
10 mmol/L Tris-HCl ( pH 7.4)
10 mmol/L NaCl
3 mmol/L MgCl2
1 mmol/L DTT
0.5 mmol/L PMSF (added before use from 100 mmol/L storage solution dissolved in ethanol)
2. Ultracentrifuge (Beckman compatible with SW28 turntable) and turntable pre-cooled to 4 °C.
3. place the tissue grinder (55 ml grinding chamber; Thomas C chamber and serrated Teflon pestle and mortar, 3431-E25). Measuring cylinder and beaker on ice.
4. Place a glass dish on ice in an ice bucket.
5. Execute the rats, remove the livers, and weigh them.
6. On a glass plate, quickly remove connective tissue with a razor blade and cut 20 g of liver into approximately 1 cm3 pieces.
7. Fill a cold beaker with 40 ml (double the volume) of lysis buffer with the liver pieces.
8. Pour approximately 30 ml of this liver fragment and buffer mixture into the pre-cooled tissue grinder chamber.
9. Attach the pestle and mortar to the propeller and slide it into the grinding chamber until it touches the buffer surface. Then hold the side of the grinding chamber and open the thruster. Gradually increase the speed of the thruster until it reaches its maximum speed while steadily advancing the chamber up the rotating grind. When the pestle reaches the bottom of the chamber, pull down on the chamber until all of the homogenate passes by the pestle and then push the chamber back along the pestle. Repeat this process 5 to 10 times and then turn off the propeller.
10. Check the efficiency of cell lysis:
(1) Place the grinding chamber on ice, remove 10 μl of lysate and dilute with 1 ml of lysis buffer.
(2) Take 2.7 μl of the diluted lysate onto a slide, add 2.7 μl of lysis buffer containing 10 μg/ml Hoechst's dye (33258 10 mg/ml; Molecular Probes or other suppliers), and lysate the cells with a 18 mm 2 The droplets were then covered with an 18 mm2 No. 1 coverslip.
(3) Compare the phase diagrams of bright DNA-stained nuclei with the same field of view.
(4) If the number of lysed cells is less than 95%, increase the speed of the propeller and continue to grind the sample 5 to 10 times.
11. Fill a funnel with 4 layers of coarse filter paper, place in a measuring cylinder buried in an ice bath, and pour the homogenate into the funnel.
12. Lysate the remaining liver fractions, filter the homogenate through the filter paper and mix with the rest of the lysate sample, recording the volume of lysate obtained.
13. Divide the lysate volume by 6 and subtract this volume from the centrifuge tube volume (35-38 ml). The volume of concentrated sucrose solution added to the 6 centrifuge tubes is determined. Add PMSF to 2 mol/L RSB solution to a final concentration of 0.5 mmol/L. Add the appropriate volume (usually 25-30 ml) to the centrifuge tubes and place on ice.
14. Add concentrated sucrose solution and an equal volume of lysate (usually 7-9 ml) to the centrifuge tube. When adding the lysate, the pipette tip should be placed against the upper part of the inner wall of the centrifuge tube so that the lysate flows down slowly, thus minimizing mixing of the lysate with the sucrose solution.
15. Place the centrifuge tube in a pre-cooled vat and equilibrate the opposite vat with lysis buffer.
16. Load the vat on the SW28 turntable and place in the pre-cooled centrifuge at 23,000 g for 30 minutes at 4°C.
17. Pipette out the lysate and sucrose solution or pour out the liquid by inverting the centrifuge tube on a beaker. Remove any residue from the inner wall with a lint-free paper towel and place the tubes on ice. Resuspend the precipitate with 2 ml of lysis buffer per tube.
18. Collect the precipitated resuspension into a 50 ml centrifuge tube (conical or round bottom), add Lysis Buffer to a final volume of 50 ml, and centrifuge at 1500 g for 5 minutes in a medical centrifuge tube.
19. Aspirate the supernatant and resuspend the nuclei in 1 ml of Lysis Buffer. Remove a small amount of sample and dilute 100-fold and count the number of nuclei on a hematocrit plate.
20. Dilute the sample to twice the desired concentration with lysis buffer, add an equal volume of glycerol, freeze in liquid nitrogen, and store at -80°C.