Mar 04, 2026

Neutrophil isolation protocol

DOI
https://dx.doi.org/10.17504/protocols.io.261ge18mov47/v1
  • Taylor P. Johnson1,
  • Valerie Escobar2,
  • Daniel W. Sirkis3,
  • Jennifer S. Yokoyama1,2,3
  • 1Fein Memory and Aging Center, Department of Neurology, Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA, USA;
  • 2Global Brain Health Institute, University of California, San Francisco, San Francisco, CA, USA;
  • 3Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, CA, USA
  • Taylor Johnson
Taylor Johnson
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DOI: https://dx.doi.org/10.17504/protocols.io.261ge18mov47/v1
Protocol Citation: Taylor P. Johnson, Valerie Escobar, Daniel W. Sirkis, Jennifer S. Yokoyama 2026. Neutrophil isolation protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge18mov47/v1
Manuscript citation:
For standard neutrophil isolation methods, please see STEMCELL Technologies, Vancouver, British Columbia, Canada, EasySep™ Direct Human Neutrophil Isolation Kit Instructions, 2025. Available from: Manufacturer website.
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: February 25, 2026
Last Modified: March 06, 2026
Protocol  Integer ID: 244130
Keywords: neutrophil isolation protocol, neutrophil isolation protocol this protocol presents method, isolation of untouched neutrophil, untouched neutrophil, native neutrophil phenotype, enriched neutrophil fraction, assisted cell separation, peripheral blood mononuclear cell, anticoagulated whole blood, cell separation, suitable for downstream nucleic acid, protein extraction, downstream nucleic acid, whole blood
Abstract
This protocol presents methods for the isolation of untouched neutrophils from whole blood drawn by standard phlebotomy using magnetic-assisted cell separation. Leveraging the STEMCELL Technologies EasySep™ Direct Human Neutrophil Isolation Kit, and its corresponding magnet, for selective targeting of non-neutrophil populations, this protocol may be inserted in the beginning of any workflow involving fresh anticoagulated whole blood, such as isolation of peripheral blood mononuclear cells (PBMC). This workflow minimizes cellular activation and preserves native neutrophil phenotype and function. The enriched neutrophil fraction is suitable for downstream nucleic acid and/or protein extraction to enable assessment of neutrophil-specific states. This protocol supplements original work published by STEMCELL Technologies, Vancouver, British Columbia, Canada.
Materials
  • 8.5 ml acid-citrate-dextrose (ACD) or ethylenediaminetetraacetic acid (EDTA) phlebotomy tube (whole blood drawn)
  • STEMCELL Technologies, Vancouver, BC, Canada. EasySep™ Direct Human Neutrophil Isolation Kit (Cat. No. 19666)
  • STEMCELL Technologies, Vancouver, British Columbia, Canada. EasySep™ Magnet (Cat. No. 18000)
  • EDTA, 0.5 M (pH 8.0), molecular biology grade, 100 ml
  • 5 ml (12 x 75 mm) polystyrene round-bottom tubes
  • 15 ml conical tubes
  • 50 ml conical tubes
  • 70% ethanol
  • Microcentrifuge tubes
  • 1x PBS (-)Ca2+, (-)Mg2+
  • Hemocytometer
  • Trypan blue
  • Tissue culture biosafety cabinet
  • Inverted brightfield microscope
  • Mechanical pipettes (p1000, p200, p20)
  • Mechanical pipette filter tips (p1000, p1000 wide-bore, p200, p20)
  • Serological controller
  • Serological pipettes (10 ml, 5 ml)
  • Waste container
  • Bleach
Protocol materials
STEMCELL Technologies, EasySep™ Magnet
EDTA
Trypan Blue
1X PBS
STEMCELL Technologies, EasySep™ RapidSpheres™
10% Bleach
STEMCELL Technologies, EasySep™ Neutrophil Isolation Cocktail
STEMCELL Technologies, EasySep™ Direct Human Neutrophil Isolation Kit
70% ethanol
Materials preparation
30m 30s
Obtain whole blood drawn into ACD or EDTA phlebotomy tube.
Clean and sanitize tissue culture hood with 70% ethanol , wipe down with Kimwipe, and UV sterilize.
Allow biospecimen to come to Room temperature (00:30:00 post-draw) and briefly spin to remove residual whole blood from the top of tube.
30m

Note
For standard neutrophil isolation methods, please see STEMCELL Technologies, Vancouver, British Columbia, Canada, EasySep‱ Direct Human Neutrophil Isolation Kit Instructions, 2025. Available from: Manufacturer website.

Spray phlebotomy tube, STEMCELL Technologies, EasySep™ Magnet , and STEMCELL Technologies, EasySep™ Direct Human Neutrophil Isolation Kit reagents into tissue culture hood with 70% ethanol .
Prepare isolation kit tubes and reagents in tissue culture hood:
Label 3x 5 mL (12 x 75 mm) polystyrene round-bottom tubes per sample as “#1”, “#2”, and “#3”.
Aliquot 2 µL of0.5 Mass Percent EDTA into round-bottom tube #1.
Note
IMPORTANT: Skip this step if beginning with whole blood collected in EDTA phlebotomy tube.

1 x 15 mL conical tube per sample.
Supplement 5 mL 1x PBS with 10 µL 0.5 Molarity (M) EDTA per sample (final concentration 1 millimolar (mM) EDTA ).
10 µL Trypan Blue (diluted 1:25 in 1X PBS ) in microcentrifuge tube.
STEMCELL Technologies, EasySep™ RapidSpheres™ , vortexed at high speed for 00:00:30 to mix.
30s
10% Bleach in waste container.
Biospecimen preparation
Carefully remove biospecimen tube rubber stopper and place upside down in tissue culture hood to minimize potential for mess.
Gently pipette total volume of whole blood into labeled 50 mL conical with 10 mL pipette tip to ensure blood is uniform prior to sampling for neutrophil isolation procedure.
Proceed with PBMC isolation or other primary phlebotomy tube application using the remaining whole blood volume.
Using a mechanical pipette with p1000 wide-bore tip, slowly remove 998 µL whole blood from the 50 mL conical tube and transfer to tube #1. Be sure to remove whole blood slowly and evenly from tip to minimize loss.
If supplementing with 2 µL of 0.5 Molarity (M) EDTA , gently pipette to mix 2-3 times with the same p1000 wide-bore tip to evenly distribute throughout sample (final EDTA concentration 1 millimolar (mM) ). Be sure to remove whole blood slowly and evenly from tip to minimize loss.
Continue with STEMCELL Technologies, EasySep™ Direct Human Neutrophil Isolation Kit in tissue culture hood.
Negative selection
10m
Add50 µL STEMCELL Technologies, EasySep™ Neutrophil Isolation Cocktail to sample in tube #1.
Add 50 µL STEMCELL Technologies, EasySep™ RapidSpheres™ to sample in tube #1.
Gently pipette to mix 2-3 times using mechanical pipette with p1000 wide-bore tip and incubate for 00:05:00 at Room temperature .
5m
Using a 5 mL pipette tip, add 2.9 mL 1 millimolar (mM) EDTA in 1X PBS and pipette gently to mix (total volume 4 ml).
Place tube #1 into the STEMCELL Technologies, EasySep™ Magnet and incubate for 00:05:00 at Room temperature .
5m
Pour tube #1 cell suspension, by inverting the magnet containing tube #1 and its contents in one continuous motion, into tube #2.
Note
Invert completely. Do not shake. Some liquid will remain behind. The cell suspension at this step may contain a significant amount of red blood cells and may continue to look similar to whole blood.

Add 50 µL STEMCELL Technologies, EasySep™ RapidSpheres™ to tube #2 containing the enriched cells.
Gently pipette to mix 2-3 times using a 5 mL pipette tip to mix and incubate for 00:05:00 at Room temperature .
Place tube #2 into the STEMCELL Technologies, EasySep™ Magnet and incubate for 00:05:00 at Room temperature .
Pour tube #2 cell suspension, by inverting the magnet containing tube #2 and its contents in one continuous motion, into tube #3.
Note
To minimize contamination, pour along a clean area of tube #2, avoiding the specific sidewall path used during the transfer from tube #1.

Place tube #3 into the STEMCELL Technologies, EasySep™ Magnet and incubate for 00:05:00 at Room temperature .
Pour tube #3 cell suspension, by inverting the magnet containing tube #3 and its contents in one continuous motion, into a labeled 15 mL conical tube.
Note
To minimize contamination, pour along a clean area of tube #3, avoiding the specific sidewall path used during the transfer from tube #2.

Aliquot 10 µL enriched neutrophil suspension into prepared Trypan Blue microcentrifuge tube for counting.

Note
Volume of suspended cells will be variable dependent on transfer efficiencies. Undiluted cell counts are intended to be informative, not conclusive.

Centrifuge the enriched neutrophil suspension at 300 x g, 00:05:00 .
5m
Remove supernatant with a p1000 with regular-bore tip and discard in 10% Bleach waste container.
Resuspend cells in appropriate buffer for downstream applications according to study goals.

Note
Intact cells are not intended to be stored at any temperature for any duration of time.

Record all observations and storage locations in secure spreadsheet or LIMS database system.
Protocol references
STEMCELL Technologies. (2025). STEMCELL Technologies EasySep‱ Direct Human Neutrophil Isolation Kit instructions for use [PDF]. STEMCELL Technologies. https://cdn.stemcell.com/media/files/pis/10000000912-PIS_01.pdf