Mar 20, 2026

Isolation of human primary neutrophils V.2

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gpzq51lzp/v2
Isolation of human primary neutrophils
  • 1Institute of Molecular Genetics, Czech Academy of Sciences, Prague;
  • 2J. Heyrovský Institute of Physical Chemistry, Czech Academy of Sciences, Prague
Kateřina Paldusová
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gpzq51lzp/v2
Protocol Citation: Nataliia Pavliuchenko, Tomáš Brdička, Kateřina Paldusová, Marek Cebecauer 2026. Isolation of human primary neutrophils. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpzq51lzp/v2Version created by Kateřina Paldusová
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: March 18, 2026
Last Modified: March 20, 2026
Protocol  Integer ID: 313543
Keywords: blood, isolation, neutrophil, neutrophils, primary, human, isolation of human primary neutrophil, human primary neutrophil, isolation
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) optionally 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 buffer - 1.5M NH4Cl (Sigma-Aldrich, #09718), 100mM KHCO3 (Sigma-Aldrich, #60339), 1mM EDTA disodium salt dihydrate (Sigma-Aldrich, #E5134) in tissue culture water, pH 7.2, filtered
Protocol materials
6% Dextran, 150mM NaCl
1X ACK buffer
RPMI+, 0.2% FBS
Phosphate buffered salineMerck MilliporeSigma (Sigma-Aldrich)Catalog #P2272
Ficoll-Paque™ PLUS density gradient mediaCytiva Life SciencesCatalog #17144002
Ammonium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #09718
Potassium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #60339
Ethylenediaminetetraacetic acid disodium salt dihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #E5134
10x ACK buffer
Water (sterile-filtered BioReagent suitable for cell culture)Merck MilliporeSigma (Sigma-Aldrich)Catalog #W3500
Dextran from Leuconostoc spp.Merck MilliporeSigma (Sigma-Aldrich)Catalog #31392
Sodium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9888
Fetal Bovine SerumMerck MilliporeSigma (Sigma-Aldrich)Catalog #F7524
RPMI-1640 MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #R5886
ROTI®Cell RPMI-1640Carl RothCatalog #9104.1
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
Preparation of reagents
In Water (sterile-filtered BioReagent suitable for cell culture)Merck MilliporeSigma (Sigma-Aldrich)Catalog #W3500 dissolve 1.5 M Ammonium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #09718 , 100 mM Potassium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #60339 , and 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #E5134 - further referred to as 10x ACK buffer

With HCl or NaOH, adjust to 7.2

Filter with 0.22µm filter
Store in the refrigerator for max 2 months, optionally aliquot per 1.5 mL
In Water (sterile-filtered BioReagent suitable for cell culture)Merck MilliporeSigma (Sigma-Aldrich)Catalog #W3500 dissolve 6 % (w/v) Dextran from Leuconostoc spp.Merck MilliporeSigma (Sigma-Aldrich)Catalog #31392 and 150 mM Sodium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9888
- further referred to as 6% Dextran, 150mM NaCl

Filter with 0.22µm filter
Store in the refrigerator
Before starting
Take all reagents out of the refrigerator, temper to room temperature
Dilute 1.5 mL 10x ACK buffer to 1X ACK buffer with Water (sterile-filtered BioReagent suitable for cell culture)Merck MilliporeSigma (Sigma-Aldrich)Catalog #W3500

Optionally, supplement 10 mL RPMI-1640 MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #R5886 with 0.2 % (v/v) adjustable heat inactivated and filtered Fetal Bovine SerumMerck MilliporeSigma (Sigma-Aldrich)Catalog #F7524
- further referred to as RPMI+, 0.2% FBS

Dextran separation
39m
transfer blood sample from EDTA vacuette tube into 50ml Falcon tube

30s
add 1 mL of Room temperature Phosphate buffered salineMerck MilliporeSigma (Sigma-Aldrich)Catalog #P2272 per 1 mL of blood sample

30s
add 1 mL of Room temperature 6% Dextran, 150mM NaCl per 1 mL of blood sample

30s
invert the tube 10 times

30s
incubate atRoom temperature for 00:30:00 without inverting the tube anymore

30m
transfer the upper phase into new 50ml Falcon tube, avoid transfer of any erythrocytes from the lower phase
1m
spin at 290 rcf, Room temperature, 00:05:00 , swing-up rotor


Equipment
centrifuge
NAME
5702
TYPE
Eppendorf
BRAND
022626001
SKU


5m
using Pasteur pipette and 2 1ml-pipette tips, discard the supernatant
1m
Erythrocyte lysis
11m 30s
gently resuspend pellet in 15 mL Room temperature 1X ACK buffer

first, resuspend in 5 mL , then, add the remaining 10 mL

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

5m
add 35 mL Room temperature Phosphate buffered salineMerck MilliporeSigma (Sigma-Aldrich)Catalog #P2272 to neutralise the lysis

15s
invert the tube 10 times
15s
spin at 290 rcf, Room temperature, 00:05:00 , swing-up rotor

5m
using Pasteur pipette and 2 1ml-pipette tips, discard the supernatant, remove as much red debris as possible
1m
Ficoll separation
37m
gently resuspend pellet in 10 mL Room temperature RPMI+, 0.2% FBS

1m
carefully load 10 mL Room temperature Ficoll-Paque™ PLUS density gradient mediaCytiva Life SciencesCatalog #17144002 at the bottom of the tube, make sure not to create any bubbles that would destroy the interface, load at the minimum speed

2m
spin at 1260 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
2m
using Pasteur pipette and 2 1ml-pipette tips, remove the rest of the supernatant
1m
gently resuspend pellet in 5 mL (adjustable) Room temperature ROTI®Cell RPMI-1640Carl RothCatalog #9104.1 (supplementing with FBS optional)

1m
count the cells manually or using automated cell counter