Flow Cytometry FAQs

Yes they are. CD8 and CD8α are widely used markers for identifying cytotoxic T lymphocytes (CTLs). CD8 plays an important role in mediating the recognition and excution funtions of CTLs. CD8α is a subunit of CD8 complex, which means the α chain, and it is quite important in the formation of the complex. CD8α chain not only participates in the binding with MHC-I molecules, but also regulates the activity of CTLs by interacting with other signal molecules. Therefore, the expression level and functional status of CD8α can be used for evaluating the activation and effection of CTLs, as well as CD8.

CD4 (domain1) is not the same as CD4, it means the first domain of CD4 protein, also known as D1 domain or Ig-likeV-type domain, so it can be used as a substitution of CD4.
CD4 (cluster of differentiation 4) protein, a surface molecule of immune cells which is often used to identify and label helper T lymphocytes (CD4+T cells), consists of four domains, including an extracellular domain, a transmembrane domain and an intracellular domain.

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.

Host refers to the species from which antibodies originate. Reactivity refers to species that have been experimentally proven to bind specifically to our antibodies.

The centrifugal force of the centrifuge does not exceed 300g, the centrifugal speed does not exceed 3, and the centrifugal speed does not exceed 1, so as to avoid cell damage.

We have no secondary antibodies against pigs and no antibodies against engineered yeast.

CD4 [SK3] and CD4 [RM4-5] are used for cytokine testing. On the one hand, because PMA stimulation causes CD4 endocytosis, CD4 clones SK3 and RM4-5 have less effect on endocytosis. On the other hand, the experimental samples for the detection of cytokines need to be fixed, and the CD4 binding domains of SK3 and RM4-5 are less affected by fixators.

Most mouse macrophage sorting uses F4/80 and CD11b to determine total macrophages, and then uses CD86 to measure M1 type and CD206 to measure M2 type. The above indexes can be used as a reference. If the cells obtained by flow sorting need to be cultured, it is recommended not to use CD206, because CD206 requires a fixed membrane breaking operation, and the cells will all die. In addition, the expression levels of M1 and M2 markers were not high, which was difficult to sort.

It is recommended that the teacher add CD45, CD11b and dead and alive dyes. On the one hand, there are many cell types in tumor samples and the proportion of macrophages is low, making it difficult to find the target group. Dead cells, on the other hand, readily ingest antibodies and probes, leading to apparent nonspecific staining. Moreover, dead cell autofluorescence is particularly strong, which can lead to enhanced background fluorescence, making it impossible to observe weak positive expression of some markers. Therefore, it is suggested that teachers eliminate the interference of dead cells by dead and alive dyes, and then circle CD45 positive to find the target cell population, and finally determine the proportion of macrophages by CD11b and F4/80.

A: The main chemical reagents used are 0.1M PBS, EDTA disodum, NEM, and a reducing agent DTT or 2-MEA, or TCEP, and 10kD ultrafiltration tube or desalination column.
Based on the characteristics of the antibody, it is assumed that the antibody is a conventional hybridoma monoclonal antibody (e.g. mouse IgG 2a,150kDa), and the antibody is reduced in a neutral buffer (e.g. PBS+ EDTA,PH7.2) with appropriate amounts of DTT or 2-MEA, which is exposed to -SH. Then, SMCC activated dye was added to the reduced antibody (according to a certain proportion), mixed at 37℃ or 4℃ overnight, and a chemical kit such as NEM (N-Ethylmaleimide) was added for about 0.5 h to complete the labeling. Determine whether to perform further purification operations based on the actual situation.
In addition, SATA or 2-thiimino-thiane hydrochloride can also be used to treat the antibody, and then add related reagents (SATA requires hydroxylamine hydrochloride, etc.) to activate -SH(sulfhydryl), and then add the SMCC activated dye to the activated antibody (according to a certain proportion), and overnight mixing reaction at 37℃ or 4℃. After adding a chemical kit such as NEM (N-Ethylmaleimide) blocking reaction can also be used, this way will not reduce the antibody, but may interfere with the antigen-antibody binding site, and the steps are more complicated.
The specific choice depends on the type and characteristics of the antibody. The use ratio of fluorescent dyes also needs to be set up for pre-experiment to explore.

The lysate is not recommended for use in poultry. The red blood cells of birds are quite different from those of humans or mice.

Yes, pyrolysis at room temperature (20℃ to 25℃).

The client is advised to use intracellular factor fixation agent, item No. E-CK-A109.

CD16/32 is a specific blocking agent, and the effect is better. Fc receptors are CD16 and D32 proteins on the cell surface, as well as CD64, but CD64 is strongly bound to FcR and can be well bound without specific antibodies.

No, it is recommended to use ultra-pure water, double steaming water, triple steaming water or deionized water dilution. After diluting the film breaker, the cell stain buffer will change the osmotic pressure and salt particle concentration of the sample itself, and eventually lead to cell deterioration or even death.

We have not tested the effect of this ACK on the viability of other cells. At present, in peripheral blood, our main research object is white blood cells. This lysate has little effect on white blood cells under normal conditions.

In this article, we recommend using FMO Combined isotype control as a negative control to set gates. However, FMO Combined isotype control is more complicated, so many experimenters will choose a simpler method and only use isotype control as a negative control to set gates. Isotype controls are used to eliminate non-specific binding of antibodies causing background staining to help find true negative cell populations.

When testing multiple samples at the same time, only one isotype control is needed. But if multiple experiments are performed, isotype controls need to be performed each time.

FMO combined with isotype control (FMO-ISO) is recommended for indicators with low expression or less obvious clusters, and only one tube is needed. Taking IFN-γ as an example, "samples of the group expected to express more IFN-γ" as the sample of FMO-ISO, adding other flow antibodies and isotype control antibodies of IFN-γ in addition to IFN-γ, this tube sample is called FMO combined isotype pairs. The other experimental steps are the same as the experimental group, such as dead or alive, fixed broken membrane, etc.

This product is a liquid and does not need to be dissolved with a solvent.

Antibodies packaged in μg need to be pre-tested to titrate the antibody. We did not do the corresponding titration, we need to do the antibody titration experiment according to the recommended dosage in the instructions, the known antibody concentration and the situation of our own cell samples to find out the optimal antibody dosage, so we cannot give the specific number of use.
In general, the number of times each antibody is used in one experiment is a single dye tube, and each full dye tube is added once (there is a biological control and experimental group), that is, each antibody needs to be added at least 3 times in one experiment.

The spontaneous fluorescence of dead cells will be very strong, if the dead cells are excluded without dead dye, the cell population along the diagonal distribution may appear in the scatter plot of the analysis results, affecting the experimental results. Therefore, in order to ensure the accuracy of the experimental results, for tissue samples other than the spleen, it is generally recommended to add dead dye to exclude dead cells. If it is a blood sample, if the sample is fresh and only a few cell surface stains are done, dead cells can also be excluded, but this requires pre-testing to make sure that the sample is not too many dead cells or will not interfere with the results.
We can analyze the dead cell population of the existing data to see whether the distribution of these cells in the fluorescent channel and the corresponding proportion are basically consistent with the living cell population. If they are consistent, we can not do dead cell staining; if they are inconsistent, it is recommended to add dead and dead dyes.

Prepare two tubes of cell flow machine, which are blank tubes of control group cells and treatment group cells. When boarding, all fluorescent channels are opened to see which channel has a positive signal, and the fluorescence of the drug is determined by judging the interference channel of the sample.

It is recommended to directly soak the living tissue in 1%BSA PBS or RPMI1640 medium or special tissue preservation solution, which can be preserved at 4℃ for 24h, and it is best to experiment as soon as possible. It is suggested that teachers do pre-experiments in advance to verify it.

The amount of antibody is mainly related to the volume of fluid and the number of cells. If the number of cells is small, the whole system can be incubated in half, which is 50ul cell suspension +2.5ul flow antibody. However, if there are very few target cells in the sample, a reduction in the number of cells may affect the results, resulting in only a small number of signals being detected.

The ACK Lysis Buffer cannot remove platelets. Because the platelet density is small, the platelets after centrifugation are in the supernatant of the sample and can be removed by discarding the supernatant.

Not recommended, because calcium and magnesium ions can inhibit enzyme activity.Although most of the enzyme reagents on the market will add EDTA to chelate calcium and magnesium ions, but it is still recommended that you do not use HBSS containing calcium and magnesium ions. In addition, if the indicators related to calcium ions need to be tested later, it is recommended to use HBSS without calcium and magnesium ions, such as the detection of calcium ion flow. If you want to test the indicators that need to stimulate activation, you should also be careful to use enzymes that do not contain EDTA, such as IFNγ produced by activated T cells (because EDTA chelates calcium ions, and calcium ions are important trace elements to ensure the activation process of T cells).

① Choose heparin anticoagulant tube; (2) Sample preparation: a. Human peripheral blood sample: dilute red blood cell lysate in advance, and use it now; B. BMC samples: The samples and reagents were rewarmed to 18~20℃ before the experiment was carried out; The ambient temperature of the experiment was controlled at 18~20℃. The collected fresh human blood was separated by PBMC within 1 h. When adding blood, avoid dispersing the stratified liquid level; Do not mix or shake the tube after adding. (3) During blood collection, the blood sample is fully shaken in the heparin anticoagulant tube to avoid blood coagulation (the storage time of the heparin anticoagulant tube is generally not more than 12h); ④ Cracking red time does not exceed 5min, it is recommended to do pre-experiment to find out the best time; ⑤ aseptic operation; ⑥ The stimulation and blocking time are suggested to be explored through pre-experiment.

① All sample tubes should be fixed and broken as in the experimental group, such as blank control, single standard group, fmo-iso; ② The fixed breaking time of each group should not be different for too long. If the sample size is large, it is recommended to operate in batches; ③ For tumor tissue samples, due to the small number of cells for detection, it is necessary to consider increasing the amount of antibodies and the number of computer time should be at least 1 million; ④ It is recommended to add 75um micron aperture filter for secondary filtration to improve the purity and content of immune cells; (5) Due to the longer stimulation blocking time, it is necessary to pay attention to aseptic operation before the end of the stimulation blocking time.

Both sequences of sample preparation can be applied. Red splitting before staining can reduce the number of cells and make the specific binding efficiency of antibody and antigen higher. Staining and then splitting red can reduce the number of centrifuges, can retain the number of cells to a greater extent, and is more suitable for samples with a small number of cells or rare samples. In addition, staining before cracking red may affect the expression of some indicators, such as CD122, CD200, CD56, CD183, IGM, etc. These indicators suggest that first crack red and then add antibodies, the staining effect will be better. Customers can choose the most appropriate method according to their own situation.

If the target of the study contains FcR (such as FcrRII/III), it is necessary to incubate anti-FcrRII/III antibody flow antibody, then incubate FcR blocking agent, and finally incubate other antibodies.

Flow assay is recommended with fresh cells. If you are in a special situation, it is only recommended that you first fix the prepared single-cell suspension with 4% paraformaldehyde for 30min, then wash it twice with PBS, and store it in the cell stain buffer (which can be replaced with 1% BSA PBS) at 4°C, and test it as soon as possible after receiving the reagent.

Subsequent experiments requiring cell culture procedures require the use of sterile consumables, such as intracellular factor testing or flow sorting experiments.

It is hardly to do so. The concentration of antibidy was well designed to have the best SINR, increasing of amount will result with high beckground signal.

Usually, it is difficult to do so. It might be able to detect the biomarker in some treatment group, but the concentration will be lower than the detection limit in more groups.

The differentiation of Th cells will only be effected by the treatment. The stimulation process is only used to make Th cells to produce enough cytokine for detection.

Both flow cytometry sorting and magnetic sorting can do so. Flow cytometry sorting can provide better purification but may effect the activity of cells; magnetic sorting can have the best activity with relatively lower purification.

The choose of stimulation regent is based on the type of sample. For example, the differentiation of RAW264.7 will be based on the treatment drug, stimulation operation, cell line source and cell conditon. You may check https://www.elabscience.cn/resource-flow_cytometry-detail-974 as reference.

All samples must follow the same operation process except antibody in order to avoid the effect of fixation and permeabilization.

Normal expression: All blood cells except platelet. Non-hematopoietic tissue; CD44 will not be expressed in normal hepatocyte; CD44 is expressed in Biliary epithelium; the exoression of CD44 could be increased when B cells and T cells were activated or differentiated to be memory cells.
Abnormal expression: Multiple myeloma cells, most lymphomas, chronic lymphocytic leukemia, tumor associated mast cells; the increasing of different splicing forms of CD44 (CD44v4、CD44v5、CD44v6、、CD44v7、CD44v7/8、CD44v10、CD44v3) may show the evolution of tumor.

It is required to incubate CD86 antibody on ice to keep the activity of cells, but it is possible to incubate CD206 in room temperature after fixation.