Jan 25, 2023

Public workspacePreparation of Nuclei Suspension from Human Musculoskeletal Tissues

DOI
dx.doi.org/10.17504/protocols.io.5qpvoy8mxg4o/v1
  • 1Institute of Sports Medicine Copenhagen, Department of Orthopedic Surgery, Copenhagen University Hospital – Bispebjerg and Frederiksberg, Copenhagen, Denmark;
  • 2Center for Healthy Aging, Department of Clinical Medicine, University of Copenhagen, Denmark.
Chloé Yeung
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DOI: dx.doi.org/10.17504/protocols.io.5qpvoy8mxg4o/v1
Protocol Citation: Chloé Yeung, Anja Jokipii-Utzon, Anders Karlsen 2023. Preparation of Nuclei Suspension from Human Musculoskeletal Tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoy8mxg4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 27, 2021
Last Modified: January 25, 2023
Protocol Integer ID: 49464
Keywords: nuclei isolation, tendon, muscle, frozen, snRNAseq,
Abstract
An adapted version of the 'Frankenstein' protocol (Luciano G Martelotto 2020. ‘Frankenstein’ protocol for nuclei isolation from fresh and frozen tissue for snRNAseq. protocols.io https://dx.doi.org/10.17504/protocols.io.bqxymxpw) to prepare single nuclei suspensions from freshly frozen human musculoskeletal tissues before nuclei sorting by flow cytometry.

Protocol described for one sample.

This protocol has been used in the following available studies:
PREPRINT: Distinct myofibre domains of the human myotendinous junction revealed by single nucleus RNA-seq. Anders Karlsen et al. https://doi.org/10.1101/2022.12.16.519020


Guidelines
  • Avoid freeze-thawing of tissues
  • Keep materials and reagents chilled
  • Keep work space RNAse free
  • All pipetting steps are performed on ice
Materials
Reagents
  • PBS, sterile
  • Penicillin-Streptomycin (5,000 U/mL) (15070063, Thermo Fisher Scientific)
  • Nuclei Isolation Kit: Nuclei EZ Prep (NUC101-1KT, Sigma-Aldrich)
  • PBS without magnesium
  • Phosphate Buffered Saline with 10% Bovine Albumin (SRE0036-250ML, Sigma-Aldrich)
  • 10x PBS (AM9625, ThermoFisher Scientific)
  • 1 M MgCl2 (M1028-10X1ML, Sigma)
  • Protector RNase Inhibitor (03335399001, Roche)
  • Trypan Blue Solution, 0.4% (15250061, Thermo Fisher Scientific)

Materials
  • Scalpel
  • 50 ml tube
  • 15 ml tube
  • Cryotube
  • 2 ml screw cap tubes
  • 2 ml microcentrifuge tubes
  • 1.5 ml microcentrifuge tube
  • 2.3 mm diameter stainless steel (11079123ss, BioSpec Products)
  • 1.0 mm silicon carbide beads (11079110sc, BioSpec Products)
  • pluriStrainer Mini 70 µm (Cell Strainer) (43-10070-40, PluriSelect)
  • pluriStrainer Mini 40 µm (Cell Strainer) (43-10040-40, PluriSelect)

Equipment
  • Liquid nitrogen
  • Freezer cabinet or dry ice
  • MP Biomedicals FastPrep-24
  • Microcentrifuge with a cooling function
  • Centrifuge that can hold 15 ml tubes and has a cooling function
  • Neubauer Improved haemocytometer

Protocol materials
ReagentNuclei EZ lysis buffer Merck MilliporeSigma (Sigma-Aldrich)Catalog #EZ PREP NUC-101
In 4 steps
Before start
  • Pre-cool centrifuges
  • Obtain ice
  • Label and pre-chill tubes on ice
Tissue Preparation
Tissue Preparation
Collect fresh tissue in a 50-ml tube containing Amount25 mL ice-cold 1X phosphate-buffered saline containing 50 U/ml penicillin and 50 µg/ml streptomycin and keep TemperatureOn ice for transport.

Using a scalpel, dissect the tissue as desired TemperatureOn ice .
Note
E.g. For hamstring tendons, use a scalpel to scrape off any muscle fibres. For muscle tissues, cut away tendon tissue.

Cut up tissue into small pieces of around 100 µm3, remove excess PBS from tissue pieces and snap freeze in cryotubes in liquid nitrogen. Store samples at Temperature-80 °C .
Note
Each tissue sample may be divided into multiple small pieces, each frozen separately.

In a freezer cabinet or on dry ice, cut each piece of frozen tissue into smaller pieces using a scalpel. Transfer ~Amount50 mg tissue into a 2 ml screw cap tube containing homogenising beads.
Note
E.g. For tendon tissue use five stainless steel balls of 2.3 mm. For muscle tissue use five stainless steel balls of 2.3 mm and an additional 1.0 mm silicon carbide bead.

Return samples to storage at Temperature-80 °C until ready for homogenisation.

Nuclei Wash and Suspension Buffer
Nuclei Wash and Suspension Buffer
Freshly prepare Nuclei Wash and Suspension Buffer (2% bovine serum albumin in PBS without magnesium, 2 mM MgCl2, 0.2 U/l RNA inhibitor). Keep TemperatureOn ice .
Note
For each sample prepare 5 ml Nuclei Wash and Suspension Buffer.

Tissue Homogenisation
Tissue Homogenisation
Pipette Amount1 mL chilled ReagentNuclei EZ lysis buffer Sigma AldrichCatalog #EZ PREP NUC-101 to each 2 ml screw cap tube containing ~50 mg cut up frozen tissue and homogenising beads.
Homogenise the tissues at 4.0 M/S for Duration00:00:20 in a FastPrep 24. Immediately after, transfer the tubes and incubate TemperatureOn ice for 5 min .

20s
Repeat homogenisation step. Immediately after, transfer the tubes and incubate TemperatureOn ice for 5 to 15 min for the bubbles to settle.
Note
The tissue may not be completely homogenised. Extra homogenisation steps could lead to the sample overheating. This step will need to be optimised for other tissue types.

Transfer the homogenate from each tube to a pre-chilled 2 ml microcentrifuge tube and add Amount500 µL chilled ReagentNuclei EZ lysis buffer Sigma AldrichCatalog #EZ PREP NUC-101 to each tube. Mix gently using a wide bore 1000 µl pipette. Incubate the tubes TemperatureOn ice and gently mix two more times using a wide bore 1000 µl pipette to help release more nuclei from the remaining tissue.
Place a 70 µm cell strainer over a pre-chilled 15 ml tube. Filter all the homogenate from the same sample. After, wash the cell strainer with Amount1.5 mL chilledReagentNuclei EZ lysis buffer Sigma AldrichCatalog #EZ PREP NUC-101 . Centrifuge at Centrifigation500 x g, 4°C, 00:05:00 .

5m
Remove as much supernatant as possible without disturbing the pellet. Pipette Amount1.5 mL chilledReagentNuclei EZ lysis buffer Sigma AldrichCatalog #EZ PREP NUC-101 to the tube. Using a wide bore 1000 µl pipette gently resuspend the pellet by pipetting up and down 10 times. Transfer the suspension to a pre-chilled 2 ml microcentrifuge tube. Centrifuge at Centrifigation500 x g, 4°C, 00:05:00 .
5m
Remove as much supernatant as possible without disturbing the pellet. Pipette 500 µl chilled Nuclei Wash and Suspension Buffer without disturbing the pellet. Incubate TemperatureOn ice for 5 min .
Add Amount1 mL Nuclei Wash and Suspension Buffer. Resuspend the pellet using an uncut 1000 µl pipette tip, gently pipetting up and down 20 times.
Place a 40 µm cell strainer over a pre-chilled 2 ml microcentrifuge tube. Filter the nuclei suspension. After, wash the cell strainer with Amount0.5 mL chilled Nuclei Wash and Suspension Buffer. Centrifuge Centrifigation500 x g, 4°C, 00:05:00 and resuspend the pellet in the desired volume of Nuclei Wash and Suspension Buffer. Keep samples on ice.

5m
Nuclei Visualisation and Counting
Nuclei Visualisation and Counting
Mix a small volume nuclei suspension with Trypan blue. E.g. 10 µl of each. Visualise on a Neubauer Improved Haemocytometer.
Note
Nuclei will be stained blue. They can be distinguished from debris by their clear outlines and bean shape.



Nuclei purification
Nuclei purification
To purify the nuclei suspension from tissue debris, e.g., collagen extracellular matrix in tendon samples, samples may be further purified by fluorescence-activated nuclei sorting or by ultracentrifugation using an iodixanol gradient (DOI: 10.1126/sciadv.abn836).