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What is Flow Cytometry?
Flow cytometry ( FC) is a fluorescence-based technique that can quickly measure the biological properties of individual cells or organelles in a fluid flow system, such as cell population counts and protein abundance. Flow cytometry combined with a cell sorter can further classify and collect specific cells or organelles from a population. This process is called fluorescence activated cell sorting (FACS). Flow cytometry plays a very important role in cell biology, apoptosis research, and immunology.
How does Flow Cytometry work?
The instrument used in flow cytometry is called a flow cytometer, often shortened to 'cytometer.' A flow cytometer consists of a fluidic system for cell processing, an optical system, and a data acquisition system which includes signal detectors and processors.
How to choose the right antibody for flow cytometry experiments?
With the ongoing advancements in flow cytometry, the variety and quantity of flow antibodies and fluorescent labeling products have increased significantly. This, combined with the growing demand for multi-color analysis protocols, has led to challenges in antibody selection and fluorescence matching. Choosing the right flow antibody has become a crucial factor for the success of flow cytometry.
Biorbyt's Tips for Selecting Flow Cytometry Antibodies
1: Determine the basic information of the antibody
- Determine the indicators to be detected, specific surface markers or intracellular markers of target cells
- Select the appropriate antibody based on the species of the test sample
- Check the antibody instructions to see if it can be used in flow cytometry experiments.
2: Determine the parameter configuration of the instrument used
- Laser: Different flow cytometers have different lasers. Common ones are 405nm, 488nm, and 635nm, which need to be determined according to the specific instrument.
- Filter: The number of detection channels determines how many indicators can be detected simultaneously. The emission wavelength of the fluorophore is related to the filter, and the selected fluorophore should be within the detectable range of the instrument.
- Instrument: The same fluorescence may be detected in different channels on different instruments, so please refer to the instrument instructions.
3: Selection of fluorescein
- If the antigen expression is weak or the grouping is not obvious, it is recommended to choose strong fluorescence, such as PE and APC.
- If the antigen expression is strong or the grouping is obvious, it is recommended to choose the most commonly used weak fluorescence such as FITC.
- The commonly used fluorescence intensity ranking is: PE>APC>PE-cy5Cy5>PercCPp-Cycy5.5>FITC
4: Multi-color fluorescence combination
- When testing multiple indicators simultaneously, several fluorescent markers are needed. The combination of flow cytometry antibody fluorescent markers is primarily determined by three factors: the number of channels the flow cytometer can detect, the number of indicators to be tested simultaneously, and the availability of fluorescent markers for the antibodies provided by the manufacturer.
5: Selection of isotype control antibodies
- Isotype control is used to eliminate background staining caused by non-specific binding of antibodies to the cell surface. It is similar to a negative control and is an essential component of flow cytometry experiments
- Isotype control is an immunoglobulin with the same species, subtype, subchain, and fluorescent label as the primary antibody. The same dose is also required. For example, if the primary antibody is anti-human CD3-FITC (mouse IgG1), the isotype control is Mouse IgG1-FITC.
- If it is a purified primary antibody plus a fluorescently labeled secondary antibody, then an isotype control antibody corresponding to the primary antibody should be selected. For example, if it is a purified CD3+PE-labeled secondary antibody, the control should be a purified isotype control + PE-labeled secondary antibody.
- Sometimes, the isotype control shows higher expression than the specific antibody. This could be due to incorrect type or dosage of the isotype control antibody. Additionally, some markers may have low expression levels or similar antigens, leading to increased non-specific binding by the isotype control. In such cases, using alternative negative controls like blank controls is recommended.
Biorbyt can provide flow cytometry detection antibodies for protein indicators in various research fields. The following is a list of recent hot-selling products. For any questions about our products, please contact us at [email protected].
Catalog # | Name | Applications |
|---|---|---|
orb500265 | FC, ICC, IHC-P, WB | |
orb182936 | FC, ICC, IHC-P | |
orb183268 | FC, IF | |
orb689307 | FC, ELISA | |
orb699840 | FC | |
orb865688 | FC, IHC | |
orb636631 | FC, ELISA, ICC, WB | |
orb69324 | FC, ELISA, IHC, WB | |
orb35415 | FC, FC, IHC-P, WB | |
orb1474824 | FC, ELISA, ICC, IF, IHC, WB | |
orb570317 | FC, IHC, WB | |
orb344403 | FC, ELISA, IF, IHC, IP, Multiplex Assay, WB | |
orb10982 | FC, IHC-P, WB | |
orb412988 | FC, ELISA, IHC, WB | |
orb18895 | FC, ELISA, IF, WB |