Flow Cytometry-Based Cell Counting

  


1. Objective

The objective of the experiment was to count and analyze different types of cells in a given sample using flow cytometry based on light scatter and fluorescence properties.

2. Principle

Flow cytometry was used to count and characterize cells as they passed individually through a laser beam. Cells were differentiated by their size (forward scatter), internal complexity (side scatter), and fluorescence intensity if stained with specific markers. Counting was achieved using volumetric analysis or by adding a known number of fluorescent counting beads.

3. Materials Used

  • Flow cytometer (e.g., BD FACSCanto)

  • Cell suspension (e.g., peripheral blood, cultured cells)

  • Fluorescently labeled antibodies (e.g., CD45, CD3)

  • Counting beads (optional)

  • PBS (phosphate-buffered saline)

  • FACS tubes

  • Vortex mixer and pipettes

4. Procedure

  1. The cell suspension was prepared and filtered to remove clumps.

  2. Fluorescent antibodies were added to label specific cell populations.

  3. The samples were incubated in the dark for 15–30 minutes.

  4. The cells were washed with PBS and resuspended.

  5. Counting beads were added if used for absolute counting.

  6. The sample was run through the flow cytometer.

  7. Data were analyzed using cytometry software to determine cell counts and population percentages.

5. Result Interpretation

  • Total cell counts and specific subpopulations (e.g., lymphocytes, monocytes) were obtained.

  • Fluorescence plots (e.g., dot plots, histograms) showed distribution and quantity of each labeled cell type.

  • Results were expressed as cells/µL or total cells/mL, with gating strategies used to ensure accuracy.

6. Applications

  • Immunophenotyping (e.g., CD4/CD8 counts in HIV patients)

  • Cancer diagnostics (e.g., leukemia/lymphoma classification)

  • Stem cell enumeration

  • Monitoring immune response or therapy

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