Frequently asked questions

MACS GMP T Cell TransAct has been developed in the context of TexMACS GMP Medium. Therefore, only this combination can ensure highest performance. Additionally, MACS GMP Recombinant Cytokines, such as IL-2, IL-7, and IL-15 are used within the manufacturing process of T cells. For the GMP-compliant transduction of T cells, an automated process has been established on the CliniMACS Prodigy, e.g., to generate CAR-T cells. To standardize this process an initial enrichment step with the CliniMACS CD4 Reagent, CliniMACS CD8 Reagent, or CliniMACS CD62L Reagent is recommended.

Pure functional-grade antibodies are sterile-filtered unconjugated antibodies containing no preservative and supplied in phosphate-buffered saline (PBS), pH 7.2. All functional-grade antibodies are tested for low endotoxin for optimal application in cell culture.
 

MACS GMP Products are manufactured and tested under a quality management system (ISO 13485) and are in compliance with relevant GMP guidelines. They are designed following the recommendations of USP <1043> on ancillary materials. For regulatory status in the USA, please contact your local representative.

Learn more about MACS GMP quality.

Pluripotent stem cells (PSCs) grown in StemMACS iPS-Brew XF, human usually have high expansion rates. If you have been using a medium that supports lower proliferation rates, you may need to adapt your splitting ratio in order to avoid overgrowth. During the first passages, closely watch the confluency of your culture an -, if necessary - remove differentiated cells either manually or using the Anti-TRA-1-60 MicroBeads, human. You should not see spontaneous differentiation beyond passage 3.

Note: The presence of Rho-associated protein kinase (ROCK) inhibitor can induce a fibroblast-like morphology, especially after single cell splitting or with small colony pieces. This is not a sign of differentiation and will disappear at larger colony size/withdrawal of the inhibitor.

All human IL-2 IS product lots are calibrated using the NIBSC standard. You will find a general advice in the data sheet. For details, please contact the Miltenyi Biotec technical support.

REAlease MicroBead Kits have been developed for positive selection of target cells from PBMCs. REAlease MicroBead Technology relies on recombinantly engineered antibody fragments instead of antibodies to label specific cell surface markers. The antibody fragments have a low affinity for cell surface epitopes. However, when the fragments are multimerized as a complex, they bind epitopes on target cells with high avidity and enable effective magnetic cell separation. REAlease Technology controls the multimer / monomer state of the fragments and thus triggers the release of monomerized antibody fragments from the cell surface after isolation. Ultimately, the isolated, positively selected target cells are free from antibody fragments and magnetic labels and are thus available for further separation steps. Of course, the negative, i.e. non-labeled, cell fraction from this separation step is also available for further separation by positive selection.

Learn more about MACS Cell Separation strategies.

Multi-parameter or sequential sorting is a combination of two subsequent separations applied to isolate cell subsets that can be distinguished from other cell types through their expression of two different markers. This includes cell types for which a specific marker has not been defined.

Learn more about MACS Cell Separation strategies.

To ensure optimal stimulation and cell growth it is important to start the culture with appropriate cell densities and medium volumes. Ideal starting conditions for human T cell expansion are 2.5×10⁶ cells/mL/cm² and for mouse T cells 1×10⁶ cells /mL/cm² for mouse T cells. For more details about cell numbers and medium volumes that are appropriate for starting cultures in commonly used formats of culture plates, dishes, and flasks, please refer to the data sheet.

We use the entries in the UniProt database (http://www.uniprot.org) as reference. Some proteins naturally exist in several isoforms, and we generally use isoform 1 in UniProt/ the isoform named “canonical” sequence as described in UniProt for the PepTivator Peptide Pool. Exceptions thereof are clearly indicated in the data sheet.

Cells stimulated with antibody-loaded Anti-Biotin MACSiBead Particles can be directly used for any downstream processing such as cytokine analysis or immunoprecipitation. Stimulated cells can also be transfected with high efficiency. Anti-Biotin MACSiBead Particles show no autofluorescence and do not need to be removed prior to flow cytometry. However, when removal of Anti-Biotin MACSiBead Particles is desired, e.g., for restimulation with the particles, this is easily achieved by using the MACSiMAG Separator.

We recommend to use two different indirect MACS MicroBeads for this approach. When performing multi-parameter sorting using the Anti-Biotin MultiSort MicroBeads for biotinylated primary antibodies, it is not possible to use a biotin-labeled antibody for the second separation as well. Since the Anti-Biotin MultiSort MicroBeads do not necessarily cover all biotin residues, it is impossible to ensure that the MicroBeads will only recognize the second biotin-conjugated antibody. For the second separation fluorochrome-coupled antibodies can be used in combination with Anti-Fluorochrome MicroBeads (e.g Anti-PE MicroBeads). 

The ED50 value and units/mg determined by an assay are only valid for that respective assay. Even minor variations in the assay conditions can result in a different activity. ED50 value and units/mg determined by different assays usually result in completely different values and cannot be compared. This can only be overcome by calibration of the assay with a defined standard, a reference cytokine with an approved activity. This standard is measured in our assay, and we adjust the assay conditions to measure exactly the activity stated by NIBSC (= assay calibration). As a result, the measured activity of our cytokines is not dependent on the assay and assay variations anymore. It should be kept in mind that activity values from different suppliers are only comparable when such a standard is used in all assays.

 It is important to calculate how many active units of cytokine are available in the product. The most precise and reliable way to add recombinant cytokine to your cell culture is to dose units/mL (U/mL). This will increase the reproducibility of your experiments as compared to ng/mL dosing. Whenever possible, it is recommended to use units that were determined by a calibrated assay, because this will give you the highest reliability for reproducible results.

In general, our MicroBeads enable separation of cells that can be identified as a distinct, positively staining population by flow cytometric analysis. It is difficult to give an exact minimum number of surface molecules required for the magnetic MicroBeads to retain the cells. The size of the cells and quality of the antibody are also very important factors.  The absolute number of surface molecules is not the only important factor for a sufficient labeling and separation. You must also use the best column type for your particular application. For more information, please contact your local technical support. 

Dissociation of brain tissue results in a large amount of dead cells and debris. In addition, removal of calcium and magnesium ions with EDTA leads to precipitation of DNA from the dead cells and cell clumping in single-cell suspensions during and after the dissociation. If this occurs, simply add more of solutions 3 and 4 as indicated on the product data sheet.

Below you find a customer protocol for the isolation of platelets by depletion of unwanted cells:

1. Start with 9.0 mL of citrate-stabilized blood.
2. Centrifuge for 10 min at 200xg at room temperature. This yields a thrombocyte-rich supernatant with 3x10⁷ erythrocytes, 1.4x10⁶ lymphocytes, and 5x10⁸ platelets.
3. Combine 350 µL of this supernatant with 100 µL of CD45 MicroBeads, human and 100 µL of CD235a (Glycophorin A) MicroBeads, human.
4. Incubate at room temperature (25 °C) for 6 min. In the mean time, prepare a MS Column and MiniMACS Separator or OctoMACS Separator.
5. Apply the sample to the MS Column.
6. Wash three times with 500 µL PBS containing 2 mM citrate and 0.5% BSA. The flow-through will contain the untouched platelets. If a higher concentration of platelets is required, use only one washing step.

The term xeno-free or xenogeneic-free (XF) refers to the absence of any “foreign” component (relative to the native species you are working with) within cell culture media formulations. A designated XF medium for human cell lines for example only contains humanderived components (like human serum). Usage of components like fetal bovine serum or growth factors from other species are not allowed.

Check the following factors:

• Did you incubate at higher temperatures than 4°C or longer than indicated in the data sheet?
• Did you use more antibody or MACS MicroBeads than indicated in the data sheet?
• Did you choose the right column? Was the column overloaded?
• Did you have clumps in your cell suspension? Filter your cells before staining with MACS MicroBeads to avoid clumps.

Production processes at Miltenyi Biotec are developed to avoid the use of animal, human or viral components. Information and review regarding the origin of all materials used for the manufacturing of Miltenyi Biotec products is part of the release process of the raw material before using the components in manufacturing. For further information please refer to our Animal Origin Policy Statement for MACS GMP Products which can be found on our website (www.miltenyibiotec.com/gmp) and the product-specific certificates of origin (CoO).

Biological activities of products from different vendors can only be compared if the cellular assays are calibrated with a defined international standard (from the NIBSC), or if the samples are
measured in parallel using the same assay, the same batch of cells, and the same set of conditions. Because even minor variations in assay conditions can result in different activity, we use the NIBSC standard whenever possible. For product-specific comparison data, please have a look at the product pages online. In addition, it is recommended to determine the optimal concentration for your specific application by a dose-response experiment, because the standardized assay for determination of the biological activity cannot comprise all possible aspects of different applications.

The frequency of dendritic cells (DCs) and DC subsets varies among different mouse strains.

In BALB/c mice, the approximate DC frequencies in the spleen are as follows:

• plasmacytoid DCs: 1% 
• CD4⁺ DCs: 0.8–1.0% 
• CD8⁺ DCs: 0.4–0.5%

Most of our MACS MicroBeads do not contain BSA. Usually, MACS MicroBeads are supplied in a solution containing a stabilizer and 0.05% sodium azide.

The anti-CD45 clone recognizes CD45.1 and CD45.2.

Ancillary/raw materials are substances used for manufacturing or extracting the active substance(s) but from which this active substance is not directly derived, such as cytokines, synthetic peptide pools, cell culture media, and activation and expansion tools.