Aug 22, 2023

Public workspaceCAMbank: CPT Field Processing v1

DOI
dx.doi.org/10.17504/protocols.io.kxygx338kg8j/v1
  • 1Weill Cornell Medicine;
  • 2BioAstra
Eliah G Overbey
Open access
DOI: dx.doi.org/10.17504/protocols.io.kxygx338kg8j/v1
Protocol Citation: Eliah G Overbey, Krista A Ryon, JangKeun Kim, Christopher E Mason 2023. CAMbank: CPT Field Processing v1. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx338kg8j/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: August 22, 2023
Last Modified: June 27, 2024
Protocol Integer ID: 86808
Keywords: CAMbank, CPT, plasma, PBMCs, astronaut, SOMA
Funders Acknowledgement:
WorldQuant Foundation
NASA
Grant ID: NNX14AH50G
NASA
Grant ID: NNX17AB26G
NASA
Grant ID: 80NSSC22K0254
NASA
Grant ID: NNH18ZTT001N-FG2
NASA
Grant ID: 80NSSC22K0254
NASA
Grant ID: NNX16AO69A
NASA
Grant ID: 80NSSC21K0316
Abstract
Field processing of CPT vacutainers for the Cornell Aerospace Medicine Biobank (CAMbank).

Instructions for preserving: Plasma, PBMCs, and RBC Pellets.
Materials
Tube Type: BD Vacutainer® Mononuclear Cell Preparation Tube (CPT) (BD Biosciences: #362753)
Perform Venipuncture
Perform Venipuncture
5m
5m
After venipuncture, invert the tubes 8 to 10 times to fully mix in the sodium heparin anticoagulant.

Store the tube upright at room temperature until centrifugation.

To ensure proper barrier formation, blood samples should be centrifuged within 2 hours of blood collection. Centrifugation more than 2 hours after specimen collection may cause incomplete barrier formation.
5m
Centrifuge Settings
Centrifuge Settings
35m
35m
Note: A swing bucket centrifuge is required.

Set centrifuge:
  • acceleration: 9
  • deceleration: 0
  • temperature: RT
  • duration: 30 minutes
  • speed: 1800xg
5m
Place the CPTs in the centrifuge.
Place a protective cover over the swing buckets in case of tube breakage.
Start the centrifuge.
Stand by the centrifuge until the centrifuge reaches max speed. Listen for signs of imbalance or compromised tube integrity.
30m
Aliquot Plasma
Aliquot Plasma
32m
32m
Carefully remove CPTs from the centrifuge and inspect for separation of red blood vs PMBCs vs plasma layers.

Transfer CPTs to a sanitized laminar flow hood.
2m
Using a P1000, carefully aliquot plasma (yellow layer above buffy layer) into 2D barcoded tubes at 500µL each.

Do not pipet close enough to disturb the buffy coat (the cloudy PBMC cell layer).

Return 2D tubes with plasma to the rack and place in the -80C freezer.
30m
Wash PBMCs
Wash PBMCs
43m
43m
Prepare the wash buffer (PBS + 2% FBS)

In a 50mL conical tube, mix:
  • 24.4mL of PBS
  • 600µL of FBS

Create more wash buffer as needed (depending on the number of tubes drawn).
5m
Add 5mL PBS + 2% FBS to each CPT tube.

Gently resuspend the peripheral blood mononucleocyte cells (PBMC) layer into the PBS + 2% FBS using a serological pipet.
3m
One CPT tube at a time, transfer the resuspended cells into a new 15mL conical tube.
3m
Add fresh wash buffer to bring the conical tube volume to 15mL.
2m
Repeat steps 8-9 for all CPTs.
10m
Pellet the PBMCs in the centrifuge.

Set centrifuge:
  • acceleration: 9
  • deceleration: 9
  • temperature: RT
  • duration: 15 minutes
  • speed: 300xg


15m
Return the 15mL conical tubes to the laminar flow hood.
Gently aspirate the media away from the cell pellet.
5m
Viably Freeze Cells
Viably Freeze Cells
15m
15m
Prepare freezing media: 10% DMSO, 90% FBS.
One CPT will require 6mL of freezing media.

3m
Resuspend the cells of each 15mL conical tube in 6mL of freezing media.
2m
Transfer 1mL of PBMCs into each cryovial.
5m
Place the cryovials into Mr. Frosty containers.
3m
Store the Mr. Frosty's at -80C overnight, then ship back to WCM for liquid nitrogen storage.
1m
Recap and freeze the CPT tubes to preserve DNA in the red blood cell pellet
1m