May 11, 2022

Public workspaceViriCan Protocol: PBMC isolation from whole blood

This protocol is a draft, published without a DOI.
ViriCan Protocol: PBMC isolation from whole blood
  • Deilson Elgui De Oliveira1
  • 1Universidade Estadual Paulista (UNESP)
Deilson Elgui De Oliveira
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Protocol Citation: Deilson Elgui De Oliveira 2022. ViriCan Protocol: PBMC isolation from whole blood. protocols.io https://protocols.io/view/virican-protocol-pbmc-isolation-from-whole-blood-b83rrym6
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: In development
We are still developing and optimizing this protocol
Created: May 09, 2022
Last Modified: May 11, 2022
Protocol Integer ID: 62289
Keywords: blood, mononuclear cells, PBMC isolation, pbmc isolation from whole blood protocol, pbmc isolation, isolation of peripheral blood mononuclear cell, whole blood protocol, peripheral blood mononuclear cell, virican protocol, requiring viable cell, viable cell, dna, cell pellet, isolation, rna
Abstract
Protocol for isolation of peripheral blood mononuclear cells for downstream applications requiring viable cells or cell pellets to extract DNA or RNA.
Guidelines
Notes:

  1. From Thermofisher (Answer Id: E4470): "Blood collected with EDTA typically has the highest DNA contamination, blood collected with heparin typically has less than that collected with EDTA, and blood collected with citrate shows the least DNA contamination of the three. (Formulation for citrate solution: 3.8% (w/v) which is 3.8 g/100 mL of water. Use 0.5 mL for every 4.5 mL of blood. Rock gently back and forth after adding citrate solution to mix.) Adding 12 µL of 5 N acetic acid per milliliter of TRIzol Reagent may help, although there may still be a problem with DNA contamination. Using plasma or serum works best. The fresher the blood sample the better the RNA. Degraded RNA has been observed in blood that has been processed in as little as two hours after drawing."

Reagent manuals:
Materials
Recommended blood collection tubes :

Lavender cap, with EDTA (Ethylenediaminetetraacetic Acid)
Green cap, with Heparin (Sodium/Lithium/Ammonium)
PBMC Separation Reagents:

ReagentFicoll Paque PLUSGE HealthcareCatalog #17144003-500 ml
ReagentHistopaque 1077Merck MilliporeSigma (Sigma-Aldrich)Catalog #1077-1
Protocol materials
ReagentFicoll Paque PLUSGE HealthcareCatalog #17144003-500 ml
ReagentHistopaque 1077Merck MilliporeSigma (Sigma-Aldrich)Catalog #1077-1
Reagent1X PBS (Phosphate-buffered saline )
Troubleshooting
Blood colection
Collect Amount4 mL of whole blood in tubes with anticoagulant*


Note
(*) Collection tubes with EDTA (lavender cap) or heparin (green cap)


PBMC isolation
Add Amount3 mL of Histopaque/Ficoll to 15mL conical centrifuge tube

Note
Bring the tubes to TemperatureRoom temperature before use


Carefully layer about Amount3 mL of whole blood on top of the Histopaque/Ficoll reagent.

Note
Let the blood layer the separation reagent by pipetting it on the wall of the conical tube with a Pasteur pipette or 1mL pipette tip.

Centrifuge TemperatureRoom temperature at Centrifigation400 x g, 00:30:00 ,

Note
Centrifugation at lower temperatures (e.g., 4°C) may result in cell clumping and poor recovery.


Carefully transfer the middle opaque phase containing the PBMC to a new identified tube as follows and proceed to obtain PBMC's lisates or viable PBMCs for cellular assays.
Note
For DNA/RNA extraction: nuclease-free 2mL microcentrifuge tube. Go to procedure starting in step 6
To isolate viable PBMC: 15mL centrifuge conical tubes. Go to the procedure starting Step 8

(Optional) PBMC lysis to extract DNA/RNA
Pellet the cells by centrifugation Centrifigation1000 x g, 4°C, 00:05:00
Note
Washing cells before addition of TRIZOL® Reagent should be avoided as this increases the possibility of mRNA degradation

5m
Add To the PBMCs pellet:

Amount250 µL of TRIZol LS Reagent

(General recommendation: Amount1 mL of TRIZol per 5-10 ×10e6 eukaryotic cells)



Homogenize to lysate the cells
Note
NOTE: According to ThermoFisher, "After homogenization and before the addition of chloroform, samples can be stored at -60 to -70°C for at least one month."

Critical
Store PBMC lisates in Trizol at Temperature-70 °C (ULT Freezer) or immediately proceed to nucleic acid isolation
Note



(Optional) PBMC clearing
10m
To the 15mL conical centrifuge tubes, add Amount10 mL of TemperatureOn ice isotonic Reagent1X PBS (Phosphate-buffered saline )

Centrifuge Centrifigation250 x g, 00:10:00

10m
Aspirate the supernatant and discard
Wash the PBMC pellet
Resuspend the cell pellet with Amount5.0 mL isotonic Reagent1X PBS (Phosphate-buffered saline )
Mix by gentle aspiration with a Pasteur pipet or pipetting up and down with 1mL tip
Centrifuge Centrifigation250 x g, 00:10:00
10m
Discard supernatant
Repeat the PBMC wash procedure twice
Proceed to downstream applications with the isolated PBMCs