Blood Sample collection and PBMC isolation JAGUAR.v4
Carla Jones, Pablo Romagnoli, Gosia Trynka
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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.
Before start
coolcell must be at room temperature
Steps
Blood Collection
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).
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
PBMC isolation
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.
Centrifuge CPT tubes at 1500g RT for 30 minutes (break and acceleration at 5)
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.
Centrifuge for 5 min at 300 RCF. Tip off supernatant without disturbing the cell pellet.
Add washing media to bring volume to 10 mL. Cap tube. Resuspend cells gently with a pipette
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.
PBMC cryopreservation
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.
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.
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
