Neutrophil visualization after whole blood isolation?

Hello Lisha van Onselen

You may check the neutrophils by May Grünwald-Giemsa staining based on their characteristic morphological appearance (for instance, there is a single nucleus, which is multi-lobed, and can have between 2 and 5 lobes).

The neutrophil pellet suspended in 100μL PBS is cytocentrifuged at 300 × g for 3 min and then fixed. The cells are spun onto the slide, stained by May Grünwald-Giemsa staining, and examined by light microscopy.

You may prepare the required reagents as follows.

Preparation of May Grunwald stain

Dissolve 0.3 g of May Grunwald dye in 100 mL absolute methanol in a 250 mL conical flask.

Warm the mixture to 50°C in a water bath for a few hours and allow it to cool to room temperature.

Stir the mixture on a magnetic stirrer and leave it stirring for 24 hours.

Filter the mixture and the stain is ready for use.

Preparation of Giemsa stain

Add 1.0 g of Giemsa dye into 66 mL of glycerol and warm the mixture in a conical flask for 1-2 hours at 50°C.

Cool the mixture to room temperature and add 66 mL of absolute methanol.

Leave the mixture to dissolve for 2-3 days, mixing it at intervals.

The stain is ready for use after you filter.

Staining protocol:

Prepare an equal volume of May Grunwald stain and phosphate buffer pH 6.8. Mix well and pour onto the slides to fully flood the slides. Stain for 10 minutes.

Prepare a 1:10 dilution of Giemsa stain with phosphate buffer pH 6.8. Mix well.

After 10 minutes of May Grunwald staining, pour away the May Grunwald stain off the slides.

Then pour the Giemsa mixture onto the slides and stain for another 15 minutes.

After 15 minutes, pour off stain and flush the slides with running tap water.

Clean excess stain with kim wipes.

Air dry the slides. You may use the hair dryer to dry the slides but use it at the lowest speed and no longer than 10 seconds each time.

Place the long cover slip on the area of interest.

The slide is now ready for examination by microscopy.

You may also want to refer to the article attached below. It may be helpful.

https://www.sciencedirect.com/science/article/abs/pii/S0242649815001194?via%3Dihub

Best.

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