Flow cytometry is a very practical technique. It is characterized by convenience and quickness, accurate results, and successful sorting of target cells. However, when it comes to the specific selection of flow cytometry antibodies, it is estimated that many small partners will have difficulties. It is not so easy to obtain a beautiful flow cytometry result graph. The most important thing to consider before the experiment is the choice of antibody and the combination of fluorescein.
What issues should be paid attention to when selecting flow cytometry antibodies?
1.Basic requirements for antibodies
a.Know clearly whether the target to be tested is intracellular or extracellular protein;
b.Select antibody reactivity in strict accordance with the source of the sample;
c.You must choose antibodies that are clearly labeled for use in flow cytometry experiments and cannot be replaced by ordinary antibodies.
2.Requirements for flow cytometry
a.Understand that the flow cytometer used has several lasers, the common lasers are 405nm, 488nm and 633nm, etc. At present, most flow cytometers are equipped with 2 lasers of 488nm and 633nm;
b.Know how many detection channels are under each laser, and the number of channels determines how many indicators can be measured at a time;
|Instrument Common||Excitation （Ex）||light Fluorescence channel||Common fluorescent label|
|flow cytometry||488nm||530/30||FITC, DyLight 488, Abfluor 488|
|633nm||660/20||APC、 Abfluor 647|
3.Choice of fluorescent label
a.If there is a direct-labeled antibody, try to choose the direct-labeled antibody as much as possible. Indirect labeling of the secondary antibody increases the experimental steps. Multiple washings will cause cell loss and low staining accuracy;
b.Weakly express the target, try to choose a strong fluorescein-labeled antibody, PE> APC> FITC> PerCP.
4.Multi-color matching scheme
a.Each channel can only use one type of label, such as FITC and AF488. The excitation and emission wavelengths of the two labels are similar, so these two labels cannot be used at the same time;
b.Minimize spectral overlap, such as PE-cy5 and APC, adjust the compensation value to be too large when both are used;
c.Weak targets are equipped with strong fluorescein. For example, the expression of CD4 in Treg cells is higher than FoxP3, so FoxP3 should use strong fluorescein-labeled antibodies;
d.It is recommended to consult the literature for five-color and above color matching, or consult the antibody manufacturer.
5.How to adjust compensation?
In multicolor flow experiments, due to the overlap of the spectra of each fluorescein, compensation adjustment is required. The following figure shows the spectra of FITC and PE fluoresceins. Near 550nm, the two dyes have spectral overlap.
The purpose of isotype control is to reduce background interference and better display the binding strength of antigen and antibody. Especially for some intracellular proteins, low expression or continuous expression proteins, the setting of isotype control is particularly necessary. Isotype control needs to select antibodies with the same species source, the same antibody subtype, and the same fluorescent label as the stained monoclonal antibody.
Recommendation: Elabscience flow cytometry antibody one-a more cost-effective overall solution for flow cytometry experiments
Elabscience has selected clone numbers reported many times in the literature as the source of flow cytometry antibodies, anti-human/rat/mouse, etc., and has a wealth of indicators, with> 1480 online indicators covering most common flow cytometry antibodies, and There are 12 kinds of fluorescent labels, which can meet the needs of many kinds of experiments. In addition, all fluorescent antibodies provide convenient Test specifications for your direct use. Most importantly, Elabscience can also customize the flow color scheme for free, so you don’t have to worry about multicolor experiments.
Product example image:
Figure six indicators are detected simultaneously, the negative and positive cell populations of all indicators can be fully separated.
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