FACS Antibodies

Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. It sorts a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell and its attached antibodies.

How to choose Antibody for FACS Experiment?

Follow this guide to accurate flow cytometry results. In a flow cytometry experiment antibodies play a crucial role. Antibodies conjugated to fluorescent markers are able to identify our specific cells of interest and quantitate the amount of our target on the cell.

There are several different production methods for antibodies, each with their positives and negatives. The antibodies differ in binding quality and ultimately the staining of the cells. Polyclonal antibodies are more likely to cross-react with proteins other than the target and are susceptible to variation between batches. Monoclonals, especially recombinant ones, are consistent and reproducible. Specifically, monoclonal antibodies bind to the same epitope, making them ideal for flow cytometry experiments. They reduce the chance of off-target binding and delivering the highest possible batch-to-batch reproducibility.

Recombinant antibodies are a serious alternative as well. Since they are genetically engineered, it is possible to change the Fc portion of the antibody, reducing or even eliminating binding to the Fc Receptors on target cells. Caution, not all desired targets are currently covered by recombinant antibodies.

When we narrow down our pool of possible antibodies to monoclonals we still have to distinguish between different clone lines. Not all are suited for flow cytometry and some even might recognize the wrong epitope, such as a product targeting an intracellular epitope when the intention is to avoid permeabilizing the cells.

One last question that remains is whether to use a directly conjugated or unconjugated antibody. Direct conjugates simplify the assay protocol, reducing the potential for mistakes to occur during sample preparation. Especially for multiplexing approaches they are the more suitable format - allowing simultaneous use of multiple antibodies raised in the same species and enabling increased experimental complexity. If the desired antibody-fluorophore combination is unavailable though, primary antibodies combined with conjugated secondary antibodies can be utilized as substitute.

Additional Reading

Fluorescent Conjugated Antibodies for FACS

Fluophore	Excitation Peak (nm)	Emission Peak (nm)	Emission Color
DyLight 405	400	421
Alexa Fluor 405	401	421
DyLight 350	352	435
Alexa Fluor 350	346	442
Atto 390	390	470
Alexa Fluor 488	493	519
FITC	492	520
Atto 488	501	523
Cy3	550	570
Alexa Fluor 594	550	570
Phycoerythrin (PE)	565	578
Atto 565	563	592
Atto 594	591	616
Atto 633	629	657
APC	650	661
Alexa Fluor 647	651	667
Cy5	650	670
PerCp	490	675
Atto 655	633	684
Cy5.5	675	694
Atto 680	684	702
Alexa Fluor 700	702	723
Cy7	778	754
DyLight 800	771	796
Alexa Fluor 790	784	814

Cell Surface Marker

Sell-surface markers automatically come to mind in the search for FACS antibodies. The antibodies are usually directed against specific surface proteins (e.g. proteins of the CD classification; CD = cluster of differentiation). After labeling, sorting can then also take place according to these characteristics.

FACS Antibodies for Immuno Oncology

Immunophenotyping is a technique used to study the protein expressed by cells. An example is the detection of tumor marker, such as in the diagnosis of leukemia. It involves the labelling of white blood cells with antibodies directed against surface proteins on their membrane. By choosing appropriate antibodies eg CD19, CD10, CD45 or HLA-DR the differentiation of leukemic cells can be accurately determined. The labelled cells are then processed in a flow cytometer. The whole procedure can be performed on cells from the blood, bone marrow or spinal fluid.

Can't find the right antibody? If you need help to find a suiting antibody for flow cytometry, please contact our customer support via phone, live chat or email.