- Materials
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- Cell Suspension Buffer (CSB): 0.35 M sucrose, 100 mM EDTA, 50 mM Tris.HCl pH 8
- Frozen tissue sample
- Consumables
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- Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
- 1 M Tris-HCl pH 8.0 (Thermo Scientific, 15893661)
- 0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
- 2.5 M sucrose
- Nuclease-free water (e.g. ThermoFisher, cat #AM9937)
- 50 ml Falcon tubes
- 5 ml Eppendorf DNA LoBind tubes
- Equipment
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- Centrifuge suitable for 5 ml Eppendorf tubes (Eppendorf centrifuge 5804/5804 R or equivalent)
- Eppendorf tube rack suitable for 5 ml Eppendorf tubes
- Scalpel
- TissueRuptor II (QIAGEN, cat # 9002755)
- TissueRuptor Disposable Probes (QIAGEN, cat # 990890)
- Florescent microscope with functionality to quantify nuclei (Logos CELENA S Digital Imaging System or equivalent)
- Heat block equipped with thermoblock suitable for 5 ml Eppendorf tubes
- 200 µm PluriStrainer® (pluriSelect, 43-50200-03)
- 100 µm PluriStrainer® (pluriSelect, 43-50100-51)
- 50 µm PluriStrainer® (pluriSelect, 43-50050-03)
- 30 µm PluriStrainer® (pluriSelect, 43-50030-03)
- PluriStrainer® Connector Ring (pluriSelect, 41-50000-03)
- PluriStrainer® Funnel (pluriSelect, 42-50000)
- P1000 pipette and tips
- 10 ml syringe
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Prepare the Cell Suspension Buffer (CSB) as follows:
Reagent Stock Final conc. Volume Tris.HCl, pH 8 1 M 0.05 M 50 ml EDTA 0.5 M 0.1 M 200 ml Sucrose 2.5 M 0.35 M 140 ml Nuclease-free water - - 610 ml Total - - 1000 ml -
Add 1 g of the frozen tissue sample to a weighing boat.
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Using the scalpel, slice the tissue into thin strips and then dice the sample.
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Transfer the tissue sample to a fresh 50 ml Falcon tube.
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Add 10 ml of the Cell Suspension Buffer (CSB) into the 50 ml Falcon tube.
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Using the QIAGEN TissueRuptor, gently homogenise the tissue sample.
- Insert the probe and pulse at minimum speed for one second. Stir the homogenate between each pulse.
- Repeat this five times.
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Assemble the pluriStrainer apparatus with a 200 μm strainer, connector ring, funnel and 50 ml Falcon tube according to the manufacturer’s instructions.
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Pass the full volume of the tissue sample homogenate through the 200 µm PluriStrainer®.
The homogenate can be gently agitated, and a small amount of negative pressure can be applied with the syringe to help pass the homogenate through the strainer.
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Disassemble the pluriStrainer® apparatus according to the manufacturer's instructions, setting aside the strained homogenate in the 50 ml Falcon tube for later use.
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Repeat the homogenisation process on any intact tissue caught by the pluriStrainer®:
Transfer any intact tissue caught by the 200 µm pluriStrainer® into a fresh 50 ml Falcon tube by inverting the strainer and tapping out the intact tissue.
Tip: A spatula can be used to help remove the intact tissue from the strainer.Add 10 ml of the Cell Suspension Buffer (CSB) into the 50 ml Falcon tube.
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Repeat steps 6-10 two more times to perform a total of three rounds of tissue homogenisation.
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Combine the contents of the 50 ml Falcon tube with the original strained homogenate set aside in step 10.
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The combined volume of 200 µm strained homogenate is ready for further processing.
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Strain the 200 µm strained homogenate through the 100 µm pluriStrainer®:
Assemble the pluriStrainer apparatus with a 100 µm strainer, connector ring, funnel and 50 ml Falcon tube according to the manufacturer’s instructions.
Pass the full volume of the 200 µm strained homogenate through the 100 µm PluriStrainer®.
Tip: The homogenate can be gently agitated, and a small amount of negative pressure can be applied with the syringe to help pass the homogenate through the strainer.Disassemble the pluriStrainer® and retain the 100 µm strained homogenate in the 50 ml Falcon tube.
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Strain the 100 µm strained homogenate through the 50 µm pluriStrainer®:
Assemble the pluriStrainer apparatus with a 50 µm strainer, connector ring, funnel and 50 ml Falcon tube according to the manufacturer’s instructions.
Pass the full volume of the 100 µm strained homogenate through the 50 µm PluriStrainer®.
Tip: The homogenate can be gently agitated, and a small amount of negative pressure can be applied with the syringe to help pass the homogenate through the strainer.Disassemble the pluriStrainer® and retain the 50 µm strained homogenate in the 50 ml Falcon tube.
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Strain the 50 µm strained homogenate through the 30 µm pluriStrainer®:
Assemble the pluriStrainer apparatus with a 30 µm strainer, connector ring, funnel and 50 ml Falcon tube according to the manufacturer’s instructions.
Pass the full volume of the 50 µm strained homogenate through the 30 µm PluriStrainer®.
Tip: The homogenate can be gently agitated, and a small amount of negative pressure can be applied with the syringe to help pass the homogenate through the strainer.Disassemble the pluriStrainer® and retain the 30 µm strained homogenate in the 50 ml Falcon tube.
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Determine the concentration of the nuclei in the purified homogenate using a fluorescent microscope and a stain appropriate for the nuclei in the sample.
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Take forward a volume corresponding to 6 million nuclei and add this to a 5 ml Eppendorf DNA LoBind tube.
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Centrifuge the 5 ml Eppendorf tube at 16,000 x g for five minutes to pellet the nuclei/cells.
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Pipette off all the supernatant and discard, taking care not to disturb the pellet.
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Add 40 µl of PBS to the 5 ml Eppendorf DNA LoBind tube.
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Thoroughly mix the tube by repeatedly flicking. Ensure the pellet breaks up and no clumps remain in the nuclei/cell suspension.
Note: You may need to flick quite hard and thoroughly to ensure the pellet breaks up and no clumps remain.