Sean Lauber:Differential cell staining

To differentially stain the cytocentrifuge smears (from BAL), the Hema3 reagent is used (Fisher, 23-123-869).

Protocol for staining (each dip is done for 1 second):

1. Dip one slide into the fixative (clearish blue) 5times.

2. Drain excess fixative.

3. Dip slide into the Xanthene solution (red) 4 times.

4. Blot onto paper towel.

5. Dip slide into the Thiazine solution (purple) 3 times.

6. Drain excess.

7. Rinse the slide immediately with distilled water.

8. Allow the slide to air dry completely overnight.

9. Mount with Permount or something similar and coverslip.

If a deeper stain is desired, add another dipping step. Remove a step is you want less staining.

Differential counting

Count up to 500 cells for each cytocentrifuge smear and classify each cell as either a macrophage/monocyte, lymphocyte, neutrophil or eosinophil. Then calculate the % for each from the total. Then you can apply the % to the total cell count to determine the total number of each of these cell types. This will let you know if certain cell types are increased upon treatment.

Macrophage/Monocyte:

- Large cells; monocytes are smaller

- Dark Staining nucleus

- Large cytoplasm that staisn clear or light purple; monocytes have a smaller cytoplasm

- Frizzled outer membrane (macrophage processes) when activated; Monocytes have a smoother outer membrane

- Can have lots of vessicles inside

- Sometimes multinucleated (when close to dividing or when recently engulfed a cell)

Lymphocyte (look very similar to monocytes so be careful):

- Small cell

- Dark staining nucleus

- Have very little cytoplasm (a tiny slit) (monocytes tend to have more) that stains clear or light purple

- Sometimes all you see is a dark staining nucleus, if you look closely you'll see the membrane containing very little cytoplasm

- Can be frizzled

Neutrophil

- Small cell

- Dark staining, lobulated nucleus

- Has little cytoplasm that stains clear or light purple

Eosinophil

- Small cell

- Dark staining, lobulated nucleus

- Has little cytoplasm with granules that stain pink

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