フローサイトメトリーと蛍光活性化細胞選別(FACS):脾臓Bリンパ球の単離
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Q1: What are the three main systems that make up a flow cytometer?
Flow cytometers consist of three integrated systems. The fluidic system transports cells in a stream so they pass in front of a laser one by one. The optical system contains lasers and detectors that recognize fluorophores. The electronic system converts collected optical data into electronic files for computer analysis.
Q2: How do fluorochrome-tagged antibodies help identify specific immune cells?
Fluorochrome-tagged monoclonal antibodies bind to known immune cell-specific epitopes on cell surfaces. Upon laser excitation, these bound fluorochromes emit a specific wavelength of light that can be detected and scored by the flow cytometer, allowing researchers to identify and distinguish different cell types based on their surface markers.
Q3: What is the difference between flow cytometry and FACS?
Flow cytometry analyzes and identifies cells based on size, granularity, and fluorescent markers. FACS extends this capability by actively sorting specific cell populations into separate containers using a vibrating nozzle that creates droplets, an electromagnet that charges cells, and an electric field that deflects them, enabling enrichment of homogenous cell populations for independent study.
Q4: How does forward scatter and side scatter help distinguish lymphocytes?
Forward scatter correlates with cell size, while side scatter is proportional to cell granularity. Lymphocytes are small cells with low forward scatter and low side scatter, which distinguishes them from larger, more granular cells. These physical parameters allow researchers to identify lymphocytes based on morphology alone before applying fluorescent antibody staining.
Q5: What gating strategy is used to isolate B lymphocytes from splenic leukocytes?
B lymphocytes are isolated through sequential gating. First, cells are gated by morphology using forward and side scatter. Then viable CD45-positive cells are selected, followed by CD45-positive leukocytes excluding CD3-positive T cells. Finally, CD19-positive cells are identified, which represent B lymphocytes. This multi-step approach ensures high purity of the sorted population.
Q6: Why is a purity control performed after FACS sorting?
A purity control verifies that the gating strategy successfully isolated the desired cell population. Sorted cells are re-analyzed using the same gating parameters to confirm that B lymphocytes comprise at least 98% of the CD45-positive cells. This quality control step ensures the sorted population is homogenous and suitable for downstream applications like gene expression analysis or adoptive cell transfer introducing donor mouse splenocytes to a host mouse and assessing success via FACS.
Q7: What is the purpose of using ACK lysing buffer during spleen cell isolation?
ACK lysing buffer is used to selectively lyse erythrocytes (red blood cells) while preserving leukocytes. After spleen tissue is dissociated and centrifuged, ACK buffer is added to the cell pellet for two minutes to remove red blood cells. This enriches the leukocyte population, allowing researchers to focus on white blood cells for subsequent flow cytometry analysis and sorting.