PE Anti-Mouse/Human CD11b Antibody[M1/70] (E-AB-F1081UD)

Q1:What are the recommendations for flow cytometry of mouse macrophages to detect M1 and M2 type macrophages?

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.

Q2:What are the markers for sorting Raw264.7 M1 and M2 cells by flow cytometry? Can we continue to cultivate after sorting?

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.

Q3:I want to test for tumor-associated macrophages, can I just use F4/80?

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.

Q4:Why centrifuge before use?

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.

Q5:What is the difference between the test-package and the weight-package of flow antibody products?

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.

Q6:What is the concentration of primary antibody?

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.

Q7:What does Isotype Control do? How to choose a suitable isotype control antibody?

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.

Q8:What does IHC mean on the datasheet? Is it a frozen tissue section? How is it different from IHC-F?

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

Q9:What auxiliary reagents are needed for staining?

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.

Q10:What are the requirements for centrifuge usage when preparing samples?

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.