Mar 04, 2026

Public workspaceNeutrophil 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 05, 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
ReagentSTEMCELL Technologies, EasySep™ Magnet
ReagentEDTA
ReagentTrypan Blue
Reagent1X PBS
ReagentSTEMCELL Technologies, EasySep™ RapidSpheres™
Reagent10% Bleach
ReagentSTEMCELL Technologies, EasySep™ Neutrophil Isolation Cocktail
ReagentSTEMCELL Technologies, EasySep™ Direct Human Neutrophil Isolation Kit
Reagent70% ethanol
Troubleshooting
Materials preparation
30m 30s
Obtain whole blood drawn into ACD or EDTA phlebotomy tube.
Clean and sanitize tissue culture hood with Reagent70% ethanol , wipe down with Kimwipe, and UV sterilize.
Allow biospecimen to come to TemperatureRoom temperature (Duration00:30:00 post-draw) and briefly spin to remove residual whole blood from the top of tube.
30m
Centrifigation

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, ReagentSTEMCELL Technologies, EasySep™ Magnet , and ReagentSTEMCELL Technologies, EasySep™ Direct Human Neutrophil Isolation Kit reagents into tissue culture hood with Reagent70% ethanol .
Prepare isolation kit tubes and reagents in tissue culture hood:
Label 3x Amount5 mL (12 x 75 mm) polystyrene round-bottom tubes per sample as “#1”, “#2”, and “#3”.
Aliquot Amount2 µL ofConcentration0.5 Molarity (M) ReagentEDTA into round-bottom tube #1.
Note
IMPORTANT: Skip this step if beginning with whole blood collected in EDTA phlebotomy tube.

Pipetting
Optional
Critical
1 x Amount15 mL conical tube per sample.
Supplement Amount5 mL 1x PBS with Amount10 µL Concentration0.5 Molarity (M) ReagentEDTA per sample (final concentration Concentration1 millimolar (mM) ReagentEDTA ).
Pipetting
Amount10 µL ReagentTrypan Blue (diluted 1:25 in Reagent1X PBS ) in microcentrifuge tube.
Pipetting
ReagentSTEMCELL Technologies, EasySep™ RapidSpheres™ , vortexed at high speed for Duration00:00:30 to mix.
30s
Mix
Reagent10% 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 Amount50 mL conical with Amount10 mL pipette tip to ensure blood is uniform prior to sampling for neutrophil isolation procedure.
Pipetting
Mix
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 Amount998 µL whole blood from the Amount50 mL conical tube and transfer to tube #1. Be sure to remove whole blood slowly and evenly from tip to minimize loss.
Pipetting
If supplementing with Amount2 µL of Concentration0.5 Molarity (M) ReagentEDTA , gently pipette to mix 2-3 times with the same p1000 wide-bore tip to evenly distribute throughout sample (final ReagentEDTA concentration Concentration1 millimolar (mM) ). Be sure to remove whole blood slowly and evenly from tip to minimize loss.
Pipetting
Optional
Critical
Continue with ReagentSTEMCELL Technologies, EasySep™ Direct Human Neutrophil Isolation Kit in tissue culture hood.
Negative selection
10m
AddAmount50 µL ReagentSTEMCELL Technologies, EasySep™ Neutrophil Isolation Cocktail to sample in tube #1.
Pipetting
Add Amount50 µL ReagentSTEMCELL Technologies, EasySep™ RapidSpheres™ to sample in tube #1.
Pipetting
Gently pipette to mix 2-3 times using mechanical pipette with p1000 wide-bore tip and incubate for Duration00:05:00 at TemperatureRoom temperature .
5m
Incubation
Pipetting
Mix
Using a Amount5 mL pipette tip, add Amount2.9 mL Concentration1 millimolar (mM) ReagentEDTA in Reagent1X PBS and pipette gently to mix (total volume 4 ml).
Pipetting
Mix
Place tube #1 into the ReagentSTEMCELL Technologies, EasySep™ Magnet and incubate for Duration00:05:00 at TemperatureRoom temperature .
5m
Incubation
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.

Critical
Add Amount50 µL ReagentSTEMCELL Technologies, EasySep™ RapidSpheres™ to tube #2 containing the enriched cells.
Gently pipette to mix 2-3 times using a Amount5 mL pipette tip to mix and incubate for Duration00:05:00 at TemperatureRoom temperature .
Pipetting
Mix
Place tube #2 into the ReagentSTEMCELL Technologies, EasySep™ Magnet and incubate for Duration00:05:00 at TemperatureRoom temperature .
Incubation
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.

Critical
Place tube #3 into the ReagentSTEMCELL Technologies, EasySep™ Magnet and incubate for Duration00:05:00 at TemperatureRoom temperature .
Incubation
Pour tube #3 cell suspension, by inverting the magnet containing tube #3 and its contents in one continuous motion, into a labeled Amount15 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.

Critical
Aliquot Amount10 µL enriched neutrophil suspension into prepared ReagentTrypan 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.

Pipetting
Centrifuge the enriched neutrophil suspension at Centrifigation300 x g, 00:05:00 .
5m
Centrifigation
Remove supernatant with a p1000 with regular-bore tip and discard in Reagent10% Bleach waste container.
Pipetting
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