Sean Lauber:Differential cell staining

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(Differential counting)
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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.
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:
+
 
 +
'''Macrophage/Monocyte:'''
- Large cells; monocytes are smaller
- Large cells; monocytes are smaller
- Dark Staining nucleus
- Dark Staining nucleus
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- Sometimes multinucleated (when close to dividing or when recently engulfed a cell)
- Sometimes multinucleated (when close to dividing or when recently engulfed a cell)
-
Lymphocyte (look very similar to monocytes so be careful):
+
'''Lymphocyte (look very similar to monocytes so be careful):'''
- Small cell
- Small cell
- Dark staining nucleus
- Dark staining nucleus
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- Can be frizzled
- Can be frizzled
-
Neutrophil
+
'''Neutrophil'''
- Small cell
- Small cell
- Dark staining, lobulated nucleus
- Dark staining, lobulated nucleus
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-
Eosinophil
+
'''Eosinophil'''
- Small cell
- Small cell
- Dark staining, lobulated nucleus
- Dark staining, lobulated nucleus
- Has little cytoplasm with granules that stain pink
- Has little cytoplasm with granules that stain pink

Revision as of 14:31, 30 October 2012

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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