Nov 12, 2024

Public workspaceNuclei Isolation and Sorting from Frozen Human Temporal Cortex_V2 V.2

DOI
dx.doi.org/10.17504/protocols.io.j8nlk4k55g5r/v2
  • 1Stephen & Denise Adams Center for Parkinson's Disease Research at Yale School of Medicine
Beatrice Weykopf
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.j8nlk4k55g5r/v2
Protocol Citation: Clemens Scherzer, Beatrice Weykopf 2024. Nuclei Isolation and Sorting from Frozen Human Temporal Cortex_V2. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk4k55g5r/v2Version created by Beatrice Weykopf
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: October 26, 2020
Last Modified: November 12, 2024
Protocol Integer ID: 106795
Keywords: nuclei isolation, frozen human temporal cortex, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000301
Abstract
This protocol is about nuclei isolation and sorting from frozen human temporal cortex.
Guidelines
This protocol need prior approval by the user's institutional review board (IRB) or equivalent ethics committee.
Materials

1. ReagentDounce tissue grinder setSigma AldrichCatalog #D8938-1SET
2. ReagentNuclei Isolation Kit: Nuclei PURE PrepSigma AldrichCatalog #NUC201-1KT
3. ReagentDTTSigma AldrichCatalog #43816-10ML
4. ReagentDPBS with no calcium and magnesiumThermo Fisher ScientificCatalog #14190-144
5. ReagentUltraPure™ BSA (50 mg/mL)Thermo Fisher ScientificCatalog # AM2616
6. ReagentRecombinant RNase InhibitorClontechCatalog #2313B
7. Reagent70 μm Sterile Cell StrainerFisher ScientificCatalog #22363548
8. ReagentCorning™ Falcon™ Test Tube with 35µm Cell Strainer Snap CapCorningCatalog #352235
9. ReagentART™ Wide Bore Filtered Pipette TipsThermo Fisher ScientificCatalog #2079G
10.ReagentFlowmi™ Cell Strainer 40 μm Bel-ArtCatalog #H13680-0040
11.ReagentDAPI Thermo Fisher ScientificCatalog #D1306



Buffer Recipes:

  1. Lysis buffer (LB):
  • Nuclei PURE lysis Buffer
  • 1:1000 DTT
  • 0.01% Triton-x-100
2. Nuclei wash and resuspension buffer (NWRB):
  • 1x DPBS
  • 0.01% BSA
  • freshly added RNase Inhibtior (40 U/ml)
3. Nuclei wash and resuspension buffer with DAPI (NWRBD):
  • 1x NWRB
  • 0.01 mg/ml DAPI
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
Prepare Buffers as described in section 'Materials'.
Method
Method
15m
15m
Cut at total of 10 sections of human temporal cortex (50 µm thickness) store on dry ice.
Transfer tissue to a glass dounce tissue grinder on regular ice.
Add Amount2 mL LB to the tissue grinder.
Homogenize tissue with pastel A 10 times and pastel B 10 times.
Transfer the homogenate into a 2 ml microcentrifuge tube.
Incubate TemperatureOn ice for Duration00:05:00 , mix with Wide bore tips once during the incubation.
5m
Incubation
Filter the homogenate with a 70 μm strainer.
Centrifuge at Centrifigation500 x g, 4°C, 00:05:00 .
Centrifigation
Remove the supernatant and resuspend the pellet in Amount1.5 mL LB with Wide bore tips.
Incubate TemperatureOn ice for Duration00:05:00 .
5m
Incubation
Centrifuge at Centrifigation500 x g, 4°C, 00:05:00 .
Centrifigation
Remove the supernatant and add Amount1500 µL NWRB to the pellet without resuspension

Centrifuge at Centrifigation500 x g, 4°C, 00:05:00 .
5m
Remove the supernatant and add Amount500 µL NWRB to the pellet without resuspension.
Add Amount1.0 mL additional NWRB to the tube and resuspend the pellet.
Centrifuge at Centrifigation500 x g, 4°C, 00:05:00 .
Centrifigation
Remove the supernatant and resuspend the pellet in Amount1.8 mL NWRBD . and filter through 40µm FLowmi cell strainer (Bel-art)
Next, DAPI-stained nuclei were sorted using FACSARIA III cell sorter (BD Bioscience)
Gating strategy:
  • use 100 µm nozzle
  • FSC-A at 5V / SSC-A at 180V gating
  • second gating is DAPI-postive nuclei using violet laser (405nm) with 450/50 bandpass filer
  • Collect approximately 90,000 events in 1.5ml eppendorf tube and count on hemocytometer for quality control



Pipetting
Immediately after collection centrifuge at Centrifigation500 x g, 4°C, 00:05:00 .
Centrifigation
Remove supernatant and resuspend pellet according to the input amount in NWRB,
For 90,000 events you resuspend in 15µl NWRB if you have less number of nuclei adjust the total volume accordingly.
Use Amount8.4 µL of nuclei (50,400 events) for 10x GEM generation and barcoding following manufacture´s protocol. Continue with GEM generation as per manufacture's instructions.
Protocol references
Habib et al 2017; pmid-28846088