PE/Cyanine7 Anti-Human CD44 Antibody[P2A1]

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.

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.

Our antibodies are concentrated, and the final concentration of the antibodies is not only related to the concentration of the antibodies themselves, but also to the abundance of the target protein in the sample. Therefore, the recommended dilution ratio for the antibodies provided in our instructions is a range, and the final dilution ratio needs to be determined through pre-experiments.You can also consult our technical team to get a recommendation of a suitable dilution ratio for your experimental conditions.

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.

IHC is an immunohistochemical experiment, and the sections used generally include IHC-P (paraffin tissue section) and IHC-F (frozen tissue section).

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.

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.

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

Because the same protein in different types of cells may have different post-transcriptional splicing bodies and post-translational modifications, such as glycosylation, ubiquitination, etc., the molecular weight is not the only constant. In addition, the protein You can find on the official Uniprot website that the molecular weights of different subtypes of proteins are different. Therefore, there will be a certain difference between the molecular weight found in the literature and the molecular weight of the antibody in the instructions. (You can check some literature references to confirm the molecular weight of the target protein in the target sample).

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.

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.

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.

The repair conditions are generally high temperature and high pressure or microwave heating repair. For example: Antigen retrieval (high pressure method): Add 10 mmol/ml citrate buffer (pH 6.0) sufficient to submerge the slices in the pressure cooker (preparation: dissolve the antigen retrieval solution in the corresponding volume of ddH2O, mix well), and heat When it boils, place the slices on a heat-resistant plastic slicing rack, put them into the pot, cover the lid, fasten the pressure valve, continue heating, set the holding pressure for 4 minutes, open the vent valve to deflate after the time is up, and the pressure returns to zero. Open the lid, take out the inner pot and let it cool to room temperature. After the solution has cooled to room temperature, take out the slices (about 30 minutes). Antigen retrieval (microwave method): Add 10 mmol/ml citrate buffer (pH 6.0) sufficient to submerge the slices in a beaker (preparation: dissolve the antigen retrieval solution in the corresponding volume of ddH2O, mix well), and heat to boiling , place the slices on a heat-resistant plastic slice rack, put them into a beaker, heat over medium-low heat for 30 minutes, take them out, leave them to cool at room temperature, and take out the slices after the solution cools to room temperature (about 30 minutes). For the selection of IHC repair solution, it is recommended to refer to the antibody instruction manual, or refer to the following scheme: for human samples, it is preferred to use EDTA repair solution with pH 9.0 (clinical samples); for rat/mouse samples, it is preferred to use citric acid with pH 6.0 Repair fluid (scientific research sample).

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 species mentioned in the antibody instructions can be confirmed, but there is no guarantee that the antibody can be used in unverified species, even if the sequence homology is high. Whether an antibody will recognize proteins in samples from other species involves many factors and we cannot predict. If there is no other choice, you must consider purchasing antibodies for use in unverified species. We recommend that you compare the immunogen sequence with your target protein sequence. The higher the homology, the greater the possibility of success. If the antibody you purchased is for use in unverified species or experimental applications, product replacement or refunds are generally not available.

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.

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 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.

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.

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 use of positive control is the basis for determining whether the antibody and detection system work normally in an experiment, and it is also a basis for determining whether the protein to be detected exists in the sample to be detected! Especially when the detected sample is uncertain whether the protein to be detected is expressed. Therefore, when there is no positive result in the experiment, it is best to choose the appropriate positive control, and most of the instructions have recommended positive controls; Or you can search according to the SwissProt or Omnigene database in the instructions, these databases often list the tissues in which the protein is highly expressed, and these samples can be used as suitable positive controls.

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.

First of all, customers need to estimate the dosage of antibody according to the antibody instructions & the results of pre-experiments. Different samples will have different antibody dilution ratios due to different antigen expression. For example, in the IHC test of an antibody, the pre-test confirmation is calculated according to the dilution ratio of 1: 50. Under normal circumstances, one slice needs to add 100μl of diluted antibody, that is, one slice needs 2μl of antibody, and 20μl can make about 10 slices.

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.

General antibody instructions will list the types of experiments for which the antibody has been verified to be suitable (such as: WB, IHC, IP, IF, etc.). If the antibody instructions do not mention the application type, it does not mean that the antibody is not suitable. For this type of analysis application, it may be that we have not done the corresponding verification, or the verification sample is not very effective. Generally, if there is no application or the reactivity is not recommended for customers, please try to follow the verified ones listed in the instructions. Type to choose the right antibody for your experiment. If customers want to try it, they can consult technical support in advance or purchase small specifications for corresponding experiments. However, if the expected results are not achieved, product replacement or refunds are generally not available.

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).