Isolation of human primary neutrophils

Nataliia Pavliuchenko; Tomáš Brdička; Kateřina Paldusová; Marek Cebecauer

Dec 05, 2024

Isolation of human primary neutrophils

DOI

dx.doi.org/10.17504/protocols.io.dm6gpzq51lzp/v1

Nataliia Pavliuchenko¹,
Tomáš Brdička¹,
Kateřina Paldusová²,
Marek Cebecauer²

¹Institute of Molecular Genetics, Czech Academy of Sciences, Prague;
²J. Heyrovský Institute of Physical Chemistry, Czech Academy of Sciences, Prague

Kateřina Paldusová

J. Heyrovský Institute of Physical Chemistry, Czech Academy ...

DOI: dx.doi.org/10.17504/protocols.io.dm6gpzq51lzp/v1

Protocol Citation: Nataliia Pavliuchenko, Tomáš Brdička, Kateřina Paldusová, Marek Cebecauer 2024. Isolation of human primary neutrophils. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpzq51lzp/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 16, 2024

Last Modified: December 05, 2024

Protocol Integer ID: 105737

Keywords: blood, isolation, neutrophil, neutrophils, primary, human

Funders Acknowledgements:

AMULET

Grant ID: CZ.02.01.01/00/22_008/0004558

GAČR

Grant ID: 23-06676S

Abstract

This protocol was designed for the isolation of human primary neutrophils from full blood or buffy coats. Its thorough optimisation was focused on minimisation of the non-specific activation and population purity. 

Image Attribution

neutrophil

Materials

○ RPMI-1640 + HEPES (Sigma-Aldrich, #R5886) supplemented with 0.2% heat-inactivated and filtered FBS (Sigma-Aldrich, #F7524)
○ colour-free RPMI-1640 (Carl Roth, #9104.1) supplemented with 0.2% heat-inactivated and filtered FBS
○ 1x PBS (Sigma-Aldrich, #P2272)
○ 6% Dextran (Sigma-Aldrich, #31392), 150mM NaCl (Sigma-Aldrich, #S9888) in Milli-Q water, filtered
○ 10x ACK - 1.5M NH4Cl (Sigma-Aldrich, #09718), 100mM KHCO3 (Sigma-Aldrich, #60339), 1mM EDTA disodium salt dihydrate (Sigma-Aldrich, #E5134) in Milli-Q water, pH 7.2, filtered

Protocol materials

Reagent6% Dextran, 150mM NaCl 
Reagent1X PBS 
Reagent1X ACK buffer 
ReagentRPMI+, 0.2% FBS 
ReagentFicoll 
Reagentcolour-free RPMI, 0.2% FBS 
Reagent1X PBS 

Safety warnings

○ work carefully not to activate the neutrophils
○ supplement the media with FBS right before use (FBS supplement optional or adjustable)
○ prepare 1X ACK right before use

Ethics statement

Prior ethics approval should be obtained before performing these experiments. This protocol requires prior approval by the user's Institutional Review Board (IRB) or equivalent ethics committee(s) for the work with human blood samples.

Before start

○ take all reagents out of the refrigerator, temper to room temperature
○ supplement the media with FBS
○ prepare 1X ACK 

Dextran separation

39m

transfer Sampleblood sample   from EDTA vacuette tube into 50ml Falcon tube

30s

add Amount1 mL   of TemperatureRoom temperature   Reagent1X PBS  per Amount1 mL   of Sampleblood sample   

30s

add Amount1 mL   of TemperatureRoom temperature   Reagent6% Dextran, 150mM NaCl  per Amount1 mL   of Sampleblood sample   

30s

invert the tube 10 times

30s

incubate atTemperatureRoom temperature   for Duration00:30:00   without inverting the tube anymore

30m

transfer the upper phase into new 50ml Falcon tube, avoid transfer of any erythrocytes from the lower phase

spin at Centrifigation290 rcf, Room temperature, 00:05:00 , swing-up rotor  


Equipment
centrifuge
NAME
5702
TYPE
Eppendorf
BRAND
022626001
SKU

using Pasteur pipette and 2 1ml-pipette tips, discard the supernatant

Erythrocyte lysis

11m 30s

gently resuspend pellet in Amount15 mL   TemperatureRoom temperature   Reagent1X ACK buffer 

first, resuspend in Amount5 mL   , then, add the remaining Amount10 mL   

incubate at TemperatureRoom temperature   for Duration00:05:00 starting from the addition of ACK  

add Amount35 mL   TemperatureRoom temperature   Reagent1X PBS  to neutralise the lysis

15s

invert the tube 10 times

15s

spin at Centrifigation290 rcf, Room temperature, 00:05:00 , swing-up rotor  

using Pasteur pipette and 2 1ml-pipette tips, discard the supernatant, remove as much red debris as possible

Ficoll separation

37m

gently resuspend pellet in Amount10 mL   TemperatureRoom temperature   ReagentRPMI+, 0.2% FBS  

carefully load Amount10 mL   TemperatureRoom temperature   ReagentFicoll at the bottom of the tube, make sure not to create any bubbles that would destroy the interface, load at the minimum speed

spin at Centrifigation1260 rcf, Room temperature, 00:30:00 , swing-up rotor (S4x750), acceleration and brake ramp: 5  
Equipment
Centrifuge
NAME
5910 R
TYPE
Eppendorf
BRAND
5943000548
SKU

Phase layout after centrifugation

30m

with a cut 1ml pipette tip or 5ml pipette tip, transfer PBMCs from the interface into new 15ml Falcon tube

using Pasteur pipette and 2 1ml-pipette tips, remove the rest of the supernatant

gently resuspend pellet in Amount5 mL (adjustable)  TemperatureRoom temperature   Reagentcolour-free RPMI, 0.2% FBS  (percentage of FBS adjustable)

count the cells manually or using automated cell counter

Public workspaceIsolation of human primary neutrophils

Isolation of human primary neutrophils