Jun 26, 2023
Blood Sample collection and PBMC isolation JAGUAR.v4
- Carla Jones1,
- Pablo Romagnoli2,
- Gosia Trynka1
- 1Wellcome Sanger Institute;
- 2Instituto Universitario de Ciencias Biomedicas de Cordoba (IUCBC)
External link: https://www.sanger.ac.uk/news_item/researchers-will-map-immune-cells-across-latin-america-with-support-from-czi/
Protocol Citation: Carla Jones, Pablo Romagnoli, Gosia Trynka 2023. Blood Sample collection and PBMC isolation JAGUAR.v4. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwjx4zlmk/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: June 01, 2023
Last Modified: June 26, 2023
Protocol Integer ID: 82762
Funders Acknowledgement:
Chan Zuckerberg initiative
Grant ID: 2021-240438
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
AIM: This protocol aims to isolate and cryopreserve PBMC from healthy donors from JAGUAR sites for multimodal single cell analysis and blood for DNA extraction for genotyping.
Image Attribution
image created in Biorender
Materials
Reagents:
- RPMI 1640 (Gibco, cat#22400-089)
- Washing media (RPMI 1% serum, 1% Lglu Lonza, Cat. #BE17-605E/U1, 1%Pen/Strep ThermoFisher, Cat. #15140122)
- Isopropanol (if using Mr.Frosty, Sigma-Aldrich, cat nº C1562-1EA) or Coolcell
- Serum (FBS) Sigma, Cat#F7524-500ml
- 2X Freezer media (each 1mL contains 800 μL RPMI and 200 μL DMSO (20%))
- Cryovial (V7634)
- BD Vacutainer CPT (CPT tube) Cat# 362753
- DMSO (Sigma)
Equipment:
- Mr. Frosty or CoolCell (CLS432001-1EA)
- Centrifuge (refrigerated swinging bucket centrifuge, e.g. Beckman Coulter Allegra X-12R)
- Ultra freezer (-80 oC freezer)
- Liquid Nitrogen tank (LN2)
- 15 mL Falcon tube (CLS430052)
- 2D barcode labels (Ziath)
Before start
coolcell must be at room temperature
Blood Collection
Blood Collection
15m
15m
Collect blood (21g needle) into the tube using the standard technique for CPT tube (3 tubes per donor). After collection, store tubes upright at room temperature (RT, 18-25oC) for up to 2hr (if longer times are required keep tubes on ice until processing).
Safety information
Work with human samples carries a risk of contamination. Needles and other sharps may expose workers to bloodborne pathogens. Consult healthy and safety personnel before using this protocol.
10m
Collect an extra 1-2mL of blood in an EDTA blood collection tube and transfer blood to a cryotube labeled with a 2D barcode for DNA extraction (whole genome sequencing) and store at -80 until processing
5m
PBMC isolation
PBMC isolation
49m
49m
After centrifugation, pipette to remove approximately half of the plasma (stored in a new tube at -80C) without disturbing the cell layer. Collect the cell layer with a Pasteur Pipette and transfer it to a 15 mL Falcon tube with a cap. *Collection of cells immediately following centrifugation will yield the best results.
5m
Centrifuge CPT tubes at 1500g RT for 30 minutes (break and acceleration at 5)
30m
Add 2mL washing media to the CPT tube to wash remaining cells and transfer to the same tube above, bring volume to 10 mL. Cap tube. Mix cells by inverting the tube slowly 5 times.
2m
Centrifuge for 5 min at 300 RCF. Tip off supernatant without disturbing the cell pellet.
5m
Add washing media to bring volume to 10 mL. Cap tube. Resuspend cells gently with a pipette
2m
Count cell numbers and viability using Trypan Blue (dilution 1:2) in a Neubauer chamber
CRITICAL: add 2D barcode stickers to the cryotubes while tube are at room temperature.
5m
PBMC cryopreservation
PBMC cryopreservation
15m
15m
Centrifuge for 5 min at 300 RCF. Aspirate as much supernatant as possible without disturbing the cell pellet. Resuspend pellets in 500 μl of 100% FCS and transfer all to a cryovial.
5m
Combine 500 μl 2X Freezer media (dropwise) with the cells in cryovial and gently pipette to mix. Place the cryovial into the Mr. Frosty (or CoolCell) and transfer to -80 oC as soon as possible.
5m
CRITICAL: cell viability will be directly correlated to the time cells spend in the presence of DMSO - the quicker the better
After 24 hrs, move cryovials into liquid Nitrogen tanks until shipping, record date of liquid nitrogen transfer
5m
Protocol references