Jun 26, 2023

Blood Sample collection and PBMC isolation JAGUAR.v4

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvwjx4zlmk/v1
Blood Sample collection and PBMC isolation JAGUAR.v4
  • 1Wellcome Sanger Institute;
  • 2Instituto Universitario de Ciencias Biomédicas de Córdoba
  • Human Cell Atlas Method Development Community
Carla Jones
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvwjx4zlmk/v1
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, Gosia Trynka, Pablo Romagnoli 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 02, 2023
Last Modified: June 26, 2023
Protocol Integer ID: 82762
Keywords: blood for dna extraction, pbmc from healthy donor, jaguar sites for multimodal single cell analysis, dna extraction, blood sample collection, pbmc, dna, jaguar site, blood, multimodal single cell analysis, healthy donor
Funders Acknowledgements:
Chan Zuckerberg initiative
Grant ID: 2021-240438
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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
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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)
  • Sterile Pasteur pipette (E1414-0310)
  • 15 mL Falcon tube (CLS430052)
  • 2D barcode labels (Ziath)
Before start
coolcell must be at room temperature
Blood Collection
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
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
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