Flow Cytometry FAQs

Yes, it is. CD44H is one of the common names for CD44. CD44 (cluster of differentiation 44) is an immune cell surface molecule, also known as Hyaluronate receptor. The CD44 protein is diverse through the presence of multiple variant shear forms (isoforms). Among them, CD44H is the standard form of CD44, which is the main variant shear form of CD44. CD44H interacts with components of the extracellular matrix, such as hyaluronan, and is involved in biological processes such as cell adhesion, migration, and signaling. CD44H is widely expressed in many cell types and has important functions in immune cells, tumor cells, and stem cells. It is important to note that different CD44 variant cut forms may have different functions and expression patterns. Therefore, in specific studies or experiments, it may be necessary to further distinguish and study specific CD44 variant shear forms (such as CD44v6, CD44s, etc.).

Yes, it is. CD3 consists of 5 different chains, namely gamma, delta, epsilon, zeta, and eta chains. Almost all anti-CD3 monoclonal antibodies target the epsilon chain, which only expressed in T cells normally.

No, they are not. CD45.1 and CD45.2 are subtype marker forms on the surface of immune cells and belong to the CD45 (cluster of differentiation 45) family. CD45 is a receptor protein tyrosine phosphatase, widely expressed in a variety of immune cells, such as T cells, B cells, macrophages and so on. CD45 exists in several subtypes, including CD45.1 and CD45.2. These two subtypes are mainly determined by different alleles on the CD45 gene. In experiments, the introduction of alleles of CD45.1 or CD45.2 can be used to distinguish between cells of different origin. The mouse strains expressing CD45.1 include FVB, RIII, SJL/J, STS/A, DA, etc. The mouse strains expressing CD45.2 included AKR, BALB/c, CBA/Ca, CBA/J, C3H/He, C57BL, C57BR, C57L, C58, DBA/1, DBA/2, NZB, SWR, 129, etc.

CD90, also known as Thy1 (thymus cell antigen 1), is a mouse immune cell surface molecule. In mice, there are two main subtypes of CD90: CD90.1 and CD90.2. The difference between the two subtypes is due to allelic differences on the loci. CD90.1 and CD90.2 are expression variants controlled by different gene alleles. For example, in C57BL/6 mice, CD90.1 expression is regulated by the Ly5.2 locus, while CD90.2 is controlled by the Ly5.1 locus. Due to the differences in the loci of CD90.1 and CD90.2, these subtypes can be used for labeling in experiments to distinguish cells of different origin. By introducing the allele of CD90.1 or CD90.2, cells can be genetically labeled to identify, trace and quantitatively analyze cells of specific origin. CD90 can be expressed on hematopoietic stem cells, neurons, all thymic cells, and peripheral T cells. The mouse strains expressing CD90.1 included AKR, BDP, MA/MyJ, etc., and more mouse strains expressing CD90.2 included Balb/c, CBA/J, C3H/He, C57BL/-, DBA, NZB/-, etc.

CD45 is a receptor-type tyrosine phosphatase, also known as cluster of differentiation 45, that affects multiple signaling pathways, including T cell receptor (TCR) and B cell receptor (BCR) by regulating tyrosine phosphorylation levels. CD45 widely exists on the surface of hematopoietic cell membrane and is expressed on all leukocyte. Six isomers have been identified according to their extracellular epitopes, while CD45RA, CD45RB and CD45RO, have been identified on the surface of human cells. Then, two new subgroups of T cells can be identified by using this isomer molecule. CD45RA+ T cells are regarded as naive T cells (Tn) which means they are not stimulated by antigen , while CD45RO+ cells are known as memory T cells (Tm) been stimulated and differentiated by antigen.

CD62L, CD62E, and CD62P are two immune cell surface molecules that belong to the selectin family. CD62L, also known as L-selectin, is a protein in the integrin family that is expressed primarily on the surface of white blood cells. It is involved in regulating the adhesion and migration of white blood cells, especially in inflammation and immune responses. By binding to its ligand, such as PECAM-1 on vascular endothelial cells, CD62L helps leukocyte to roll out of the circulation and migrate specifically to areas such as inflammation sites or lymph nodes. CD62P, also known as P-selectin, is a adhesion molecule that is expressed primarily on endothelial cells and platelets. In the process of inflammation and thrombosis, CD62P on endothelial cells can be expressed rapidly and bind to ligands (such as PSGL-1) on white blood cells to promote mutual adhesion and activation of white blood cells and platelets. CD62E, also known as E-selectin, is an adhesion molecule that is expressed primarily on endothelial cells.

The difference between these two antibodies is the clone number, which shows the particular cell line for screening antibody. Antibodies with same clonal number target exactly the same epitope. Both of these two antibodies are avaliable.

As a recommandation, F4/80 and CD11b are always used for identyfing macrophages of mouse, and CD86 and CD206 can be used for a further seperation for M1 abd M2 respectively. Specific indicators can be determined according to the literature and experimental requirements.

For the identification of mouse NK cells, CD3-CD49b+ or CD3-NK1.1+ should be selected according to the mouse strain, such as CD49b(DX5) for BALB/c mice and NK1.1 for C57BL/6 mice. CD49b is recommended for other strains.

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.

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.

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).

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.

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.

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.