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 erythrocyte, ACK lysis buffer (E-CK-A105) is needed;
For cells that are rich in Fc receptors, such as macrophages, Fc receptors blocking is necessary before staining with flow Antibody to reduce the non-specific signal. At present, we can provide human and mouse blocking agent, E-AB-F1236A Purified Anti-Human CD16 Antibody[3G8]. 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 indexes, a fixation & permeabilization kit is needed (E-CK-A109) ; and for the detection of intranuclear indexes, 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, NK cells. Fc receptors can be expressed on macrophages, dendritic cells, NK cells, etc. In the process of antibody staining in flow assay, Fc segment of FCM antibody will bind to Fc receptors on cell surface, end up with non-specific staining and lead to false positives signals. Antibodies can be incubated directly after blocking without washing.
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0.5-1 μg (1 μl-2 μL) of blocking antibody was added to 100 μL of cell suspension (containing 1x10^6 cells) and incubated at room temperature for 10 minutes. For Fc receptor-rich cells such as macrophages, the recommended dosage can be up to 2μL.
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Cell staining buffer is mainly to maintain cell vitality, keep cells stable from damagimg and operating, maximize the intensity of the fluorescence signal of ph-sensitive fluorescent dyes, reduce the non-specific binding of antibodies to target cells, chelate metal cations to reduce adhesion between cells.
Cell staining buffer might be replaced by PBS solution of 1%BSA. About 1.5 mL of cell stain buffer is required for each sample. -
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 necessart as a gating helper especially for the indicators with low expression or continuous expression.
Single color control: In a multicolor experiment, single color control is needed to adjust the fluorescence compensation if there is interference between the different channels.
FMO control: FMO control, also known as fluorescence reduction control, refers to the multi-color experiment. FMO control is applied to observe the comprehensive effect of all related fluoresents to the target channel by removing the correspinding signal. -
Isotype control antibodies are used as the basis for determining negative and positive cells. It is necessart as a gating helper especially for the indicators with low expression or continuous expression.
The Isotype control was purified from the serum of non-immunized animals, it should be the same species source, same immunoglobulin and subtype, same fluorescein label, same dose and concentration as the stained monoclonal antibody. -
The usage of test-package antibodies is well designed and verified, there is no need for an extra dilution. The usage amout of 1 test is 5 μL of antibodies per 100μL cell suspension (containing 1x10^6 cells).
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The usage of test-package antibodies is well designed and verified there is no need for an extra dilution before use. The weight-package antibody has a higher concentration, and requires a titration process for a suitable usage amount.
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The amount of antibody is related to the incubation system, if the cell suspension volume is still 100 μL, the amount of antibody remains the same. Researchers can reduce the amount to save antibody in low cell number condition by reducing the cell suspension volume.
It is recommended to use the recommended number of cells for the experiment, if the number of cells is too large, it will lead to insufficient antibody dosage, resulting in false negative; if the number of cells is too small, especially when detecting intracellular or intranuclear indicators, a large number of centrifugation operations will lose a lot of cells, resulting in insufficient number of cells for 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 will 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 the loss of antibodies.
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Host refers to the species from which antibodies originate. Reactivity refers to species that have been experimentally proven to bind specifically to our antibodies.
