How to Count White Blood Cells in Each Counting Chamber
Monocytes are a type of mammalian white blood cell that can be activated to secrete many proteins known as cytokines. These perform diverse activities such as bacterial cell recognition and signaling to other immune cells. Flow cytometry is used to study the characteristics and function of these cells for biological research, medical diagnostics and pathology. The processing of blood for analysis by flow cytometers is a highly technical procedure which should only be performed by trained and licensed or certified individuals. Due to the requirement for extensive scientific expertise, beginners should perform these methods only when appropriately supervised.
Things You'll Need
- Paraformaldehyde or other fixative
- Phosphate-buffered saline
- Distilled water
- Laboratory tools and consumables (pipettes, filter tips, tubes, etc.)
- Laboratory machines (tabletop centrifuge, magnetic stirrer, boiling plate, chemical fume hood, etc.)
- Personal protective equipment
- Peripheral blood or isolated monocytes
- Biohazard disposal system
Instructions
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Fixation of Peripheral Blood-Derived Monocytes
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1
Prepare the fixative. Paraformaldehyde is the most commonly used fixative although others such as methanol are also used. 4% paraformaldehyde is made by dissolving 4 grams of the powder in 100 milliliters of distilled water or phosphate-buffered saline, at 60 degrees Celsius on a magnetic stirrer. Adjust the pH of the solution to 7.2 with hydrochloric acid and freeze in small portions (10 to milliliters). This should be filtered before use.
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2
Ensure that fresh blood is used for the isolation of monocytes, since this affects the quality of the cells and therefore the success of downstream experiments. Isolate peripheral blood mononuclear cells, which contain the monocytes, by the technique of Ficoll-Paque density centrifugation.
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If monocytes are to be studied in their activated state, stimulate them according to an optimized protocol that is specific for the desired cytokine and immune reaction and that is routinely used in the laboratory. Follow this with a staining procedure to detect the monocyte. The stimulated or unstimulated cells are now ready for fixation.
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4
Fixation is started by centrifuging a small volume of the cells to form a pellet which is then gently resuspended in the paraformaldehyde solution. Note that different concentrations of fixative, between 1% to 4%, should be used to test the morphology and staining efficiency of the process. This range is used because of the variability in cell integrity and quality, and a stronger fixative (higher percentage) can destroy some surface markers used to recognize the staining agents. Fixation can be performed at either room temperature or on ice at 4 degrees Celsius, and this should also be experimented with to see which temperature suits the cells. Fixation should proceed for 10 to 15 minutes, and the cells can then be pelleted and rinsed in an appropriate solution (such as phosphate-buffered saline).
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For flow cytometry, the fixed monocytes should be resuspended in an appropriate volume of buffer, which is determined by the number of cells fixed and the parameters operated by the flow cytometry facility. It must be protected from direct light and kept cold in a refrigerator or a full ice bucket, especially if they have been stained prior to fixation.
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Tips & Warnings
This method is typically used by scientifically trained operators, and should not be attempted by laymen due to the complexity of handling medical machinery and toxic chemicals. Seek a demonstration and assistance from qualified scientists or technicians before proceeding, and adhere to all local and state regulations pertaining to contact with biohazardous materials and biotechnological equipment (e.g., laser flow cytometers). The user should also ensure that they have been given permission (e.g., licenses) from local or state authorities to carry out such experiments, handle biohazard waste and so on.
Paraformaldehyde is extremely toxic and the user must handle this reagent in a chemical fume hood or other such containment unit, and all tools used to handle it, such as weighing balances and utensils, must be decontaminated prior to their removal from the containment unit. It is also volatile and boiling must be avoided.
