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How to Purchase the Flow Cytometry Antibodies

Source: Elabscience®Published: Jan 17,2024

During Flow Cytometry experiments, the labeling effect of fluorescent antibodies on single-cell suspensions directly affects the quality of experimental data. Therefore, when selecting Flow Cytometry antibodies, it is necessary to comprehensively consider various factors that affect the quality and detection effect of the antibodies, such as the specificity of the antibodies, the strength of the fluorescence signal, the method of fluorescence labeling, and Isotype control, etc.


How to choose the appropriate Flow Cytometry antibodies?

1.The selection of Flow Cytometry antibodies must first meet the most basic conditions: target protein recognition specificity, reaction species, and application experiments.

2.There are two methods for fluorescence labeling of Flow Cytometry antibodies: direct labeling and indirect labeling. During the flow cytometry experiment, it is recommended to minimize the number of experimental procedures to ensure the authenticity and accuracy of the experiment. Therefore, if conditions permit, it is recommended to use directly labeled antibodies for the experiment as much as possible.

3.Selection of Flow Cytometry antibody fluorescent labeling: Different fluorescence markers have different intensities on different instruments. Taking the FACS Calibur as an example: PE>APC>PE Cyanine 5>PerCP Cyanine 5.5>FITC>PerCP. Generally speaking, PE is the strongest and suitable for weakly expressing antigens; FITC is weak and suitable for strong antigen expression, with a wide range of applications. If a single indicator is detected in the experiment, users can choose based on the abundance of target protein expression detected, while also considering comprehensive factors such as the price of labeled antibodies. If multiple indicators are detected simultaneously, it is necessary to first confirm how many channels can be detected by the Flow Cytometer, because each channel can only select one type of fluorescence, and the fluorescence between each channel can be freely matched. Avoid selecting fluorescent markers for the same channel for all indicators to prevent fluorescence overlap and mutual interference, which may affect the final experimental results. Therefore, the more channels a Flow Cytometer has, the more surface/intracellular markers can be performed simultaneously for the same sample. Common fluorescent markers include FITC, PE, PE Cyanine 5, PE Cyanine 5.5, APC, etc.


Selection of Isotype controls

One of the differences between flow cytometry and other antibody related experiments is the need to select an Isotype control. Isotype control is used to eliminate background staining caused by non-specific binding of antibodies to cell surfaces, equivalent to a negative control in experiments. Isotype control antibody requires selecting antibodies with the same origin, subtype, and fluorescent labeling as the labeled antibodies. For example, for PE labeled human CD3 antibody, if the antibody is derived from mice and the subtype is IgG2a, the PE labeled mice IgG2a must be selected as the Isotype control.


Precautions for using Flow Cytometry antibodies

1.It is recommended to use an appropriate proportion of cells and antibodies in the experiment. Excessive cells or antibodies may affect the experimental results, so it is recommended to optimize the experimental conditions before the experiment.
Note: Different Flow Cytometry techniques have significant differences in the demand for antibodies, for example, traditional Flow Cytometry (using a sheath fluid flow system) requires 1×106 cells are the initial sample loading amount, and the antibody dosage is 10 μL. Microflow Cytometry only requires 5×105 cells, corresponding antibody dosage can be reduced to 5 μL.

2.Flow Cytometry antibodies labeled with fluorochrome should be stored at 4 ℃ in a dark place and not frozen.

3.Try to choose Flow Cytometry antibodies that have been validated through experiments to ensure the experimental results. Elabscience® Flow Cytometry antibodies have been experimentally validated and relevant data graphs can been provided for user reference.


Related references

Flow Cytometry (FCM) is a technique for rapid quantitative analysis and sorting of cells or other biological particles (such as microspheres, bacteria, small model organisms, etc.) arranged in a single column in a fluid flow. Flow Cytometry emerged in the 1960s and 1970s and has the advantages of fast speed, high precision, and good accuracy. It is a very advanced cell quantitative analysis technology. This technology is widely used in multiple fields such as cell biology, immunology, hematology, oncology, pharmacology, genetics, and clinical testing, such as detection of cell cycle/apoptosis/cell viability, study of the relationship between lymphocyte subsets and diseases, immune monitoring after organ transplantation, detection of tumor specific indicators, study of drug action mechanisms, screening of new drugs, and so on.