Flow Cytometry
Flow Cytometry FAQs
In order to provide better customer support and address customers' questions about FCM antibodies, we have compiled and published answers to the frequently-asked questions (FAQs) about FCM antibodies, which will be continuously updated.
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For samples with erythrocytes, ACK lysis buffer (E-CK-A105) is needed;
For cells that are rich in Fc receptors, such as macrophages, Fc receptor blocking is necessary before staining with flow antibody to reduce the non-specific signal. At present, we can provide human and mouse blocking agents, E-AB-F1236A Purified Anti-Human CD16 Antibody[3G8] and E-AB-F0997A Purified Anti-Mouse CD16/32 Antibody[2.4G2].
Cell staining buffer (E-CK-A107) is required in the process of cell staining.
For the detection of intracellular markers, a fixation & permeabilization kit is needed (E-CK-A109); and for the detection of intranuclear markers, a specific staining kit (E-CK-A108) is required.
Dead cell dyes are also used for flow cytometry of tissue samples such as tumors. -
Blocking process is required when detecting macrophages, dendritic cells, and NK cells. Fc receptors can be expressed on macrophages, dendritic cells, NK cells, etc. In the process of antibody staining in flow assay, the Fc segment of the flow antibody will bind to Fc receptors on the cell surface, ending up with non-specific staining and leading to false positive signals. Antibodies can be incubated directly after blocking without washing.
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Blank control: used to set the voltage of each channel.
Isotype control: Isotype control antibodies are used as the basis for determining negative and positive cells. It is necessary as a gating helper especially for the markers with low or continuous expression.
Single color control: In a multicolor experiment, a single color control is needed to adjust the fluorescence compensation if there is interference between different channels.
FMO control: FMO control, also known as fluorescence-minus-one control, refers to the multicolor experiment. FMO control is applied to observe the comprehensive effect of all related fluorescents on the target channel by removing the corresponding signal. -
Isotype control antibodies are used as the basis for determining negative and positive cells. It is necessary as a gating helper especially for the markers with low or continuous expression.
The isotype control is purified from the serum of non-immunized animals; it should be of the same species source, same immunoglobulin class and subtype, same fluorochrome label, and the same dose and concentration as the stained monoclonal antibody. -
The usage of test-package antibodies is well designed and verified, so there is no need for an extra dilution. The usage amount of 1 test is 5 μL of antibodies per 100 μL cell suspension (containing 1×10⁶ cells).
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The usage of test-package antibodies is well designed and verified, so there is no need for an extra dilution before use. The weight-package antibody has a higher concentration and requires a titration process to determine a suitable usage amount.
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The amount of antibody required depends on the incubation system. If the cell suspension volume remains at 100 µL, the antibody amount stays unchanged. To conserve antibody when working with low cell numbers, researchers can proportionally reduce the cell suspension volume.
It is advisable to use the recommended cell number for the assay. Excessively high cell numbers may lead to insufficient antibody binding and yield false-negative results. Conversely, too few cells—especially when detecting intracellular or nuclear markers—can result in substantial cell loss during repeated centrifugation steps, leaving insufficient cells for the final detection. -
The stability of the flow antibody can be different from each other based on differnet fluorescein. It is recommended to verify the freeze-thawed antibody through pre-experiments before determining. However, it is better to avoid the freeze-thawed cycling in any FCM antibody.
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During the transportation of antibodies, antibodies may stick to the tube wall or cap due to turbulence. So after receiving the antibodies, moderate centrifugation will collect the antibodies on the tube wall or cap to the bottom of the tube to avoid loss of antibodies.
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Host refers to the species from which antibodies originate. Reactivity refers to the species that have been experimentally proven to bind specifically to our antibodies.
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The centrifugal force of the centrifuge does not exceed 300×g, the centrifugal speed does not exceed 3, and the centrifugal speed does not exceed 1, so as to avoid cell damage.
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When testing multiple samples at the same time, only one isotype control is needed. But if multiple independent experiments are performed, isotype controls need to be included each time.
