JNAR Protocol

RNA delivery into Primary Human Neutrophils by Electroporation

Núria Crua· Postdoc· University of Lausanne (UNIL)

Published Jul 27, 2026

~1h 21m total· 0 contributions

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JNAR-ID: JNAR-72302191

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PublishedLast updated Jul 27, 2026

Objective

This protocol describes the isolation of primary human neutrophils from peripheral blood and their subsequent treatment with RNA by electroporation for downstream functional analyses.

Classification

Sample typeRNACells

TechniqueTransfection

Experimental contextEx vivo

Biological modelPrimary CellsHuman

Reproducibility status

Not reproducible

1 step blocks the protocol — it can't be completed end-to-end until it is fixed.

21 healthy1 blocking

Step 21Severe loss of cell viability after electroporationGo to step

⏱ Total 1 h 21 min2 sections22 steps

Neutrophil Isolation

31 min

Put the same volume of "Polymorphoprep" as your blood sample in a falcon tube, 9 mL of Polymorphoprep for sample.

Layer the blood sample by pipetting carefully over the "Polymorphoprep" while holding the tube almost horizontally.

Critical stepLayering must be gentle to avoid mixing layers and ensure clean separation of neutrophils.

Centrifuge the tube with the lowest setting for acceleration and deceleration for 20 minutes at 1800 rpm at RT (approximately 25 minutes).20 min

Remove the neutrophils with a pipet and put them in a falcon tube.

Critical stepCareful aspiration is required to avoid disturbing the interphase and contaminating the neutrophil layer with other cell types.

Wash the neutrophils with PBS by centrifuging them for 5 minutes at 1600 rpm at RT and rejecting supernatant.5 min

Add 1 ml of ACK to the tube to lyse the remaining red blood cells for 1 minute.1 min

Wash the neutrophils with PBS by centrifuging them for 5 minutes at 1600 rpm at RT and rejecting supernatant.5 min

Re-suspend the cells in 1 ml RPMI + FCS.

Count the cells in a 1:10 dilution.

9.1

Record donor, counts (×10⁶/mL), and viability (%) in the table below.

Cell Counting Table

Donor	Counts (×10⁶/mL)	Viability (%)
1
2

Electroporation

50 min

Try to have 1×10⁷ cells per condition and add the desired quantity of RNA.

Incubate for 20 minutes on ice.20 min

Centrifuge all tubes at 500 g for 5 minutes at 4°C.5 min

Remove supernatant and add 200 µl of Opti-MEM media without FBS or prepared DPBS with Mg²⁺/Ca²⁺.

Centrifuge all tubes at 500 g for 5 minutes at 4°C.5 min

Remove supernatant and add 200 µl of Opti-MEM media without FBS or prepared DPBS with Mg²⁺/Ca²⁺.

Add the corresponding volume of circRNA and mix properly.

Critical stepMixing must be gentle but thorough to ensure uniform RNA distribution without damaging cells.

Incubate for 20 minutes at 4°C.20 min

Transfer cells into 2 mm gap electroporation cuvettes.

Critical stepCells must be transferred quickly and without bubbles to avoid arcing and cell death during electroporation.

Set electroporator program 1: 230 V, 950 µF, infinity, 1 mm.

Critical stepCorrect program settings are essential for successful electroporation; incorrect settings can kill cells or prevent RNA delivery.

TipCheck that a small 'p' is visible in the bottom left corner of the machine display before pulsing.

Place the cuvette and press the green Pulse button. The machine should display a value around 30.

Critical stepPulse must be delivered immediately after placing the cuvette to prevent cell settling and ensure uniform field exposure.

Immediately resuspend the cells in 1 mL of complete media containing FBS + IL2 (for T cells) or complete medium (for neutrophils).

Critical stepRapid resuspension in complete media is critical to recover cells and prevent apoptosis after electroporation.

BlockerSevere loss of cell viability after electroporation

Viability was reduced to less than 10% after electroporation. This outcome prevented further analysis and suggests the electroporation parameters or cell handling are incompatible with primary human neutrophils.

Reported by Núria Crua · Jul 27

Count the cells to check numbers and viability.