Frequently asked questions

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.

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.

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.

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.

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). 

 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.

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.

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.

The autoMACS Running Buffer contains BSA, which will degrade if the solution is heated above 42 °C. For this reason it should not be autoclaved. 

For preparing a sterile MACS Separation Buffer, we recommend diluting the MACS BSA Stock Solution 1:20 with autoMACS Rinsing Solution. Use unopened bottles and prepare the solution under sterile conditions (e.g., under a laminar flow hood).

Alternatively, you can prepare the buffer from scratch using autoclaved PBS (can be autoclaved with EDTA) and BSA from a sterile-filtered stock solution, or by passing a fully prepared solution through a sterile bottle-top filter. Keep in mind that concentrated BSA stock solutions tend to clog sterile filters, so the BSA should be well-diluted prior to filtering.

Pure antibodies are unconjugated antibodies supplied in buffer containing stabilizer and 0.05 % sodium azide. They are usually required for applications such as analysis of cellular markers using flow cytometry or immunofluorescence. 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.

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. 

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.

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.

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.

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.

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.

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.

We recommend to follow our detailed application protocols for the isolation and cultivation of highly purified and viable microglia.
Isolation and cultivation of microglia from neonatal mouse or rat brain
Isolation and cultivation of microglia from adult mouse or rat brain

Red blood cell lysis is not necessary when doing a separation, unless it is mentioned in the data sheet. Red blood cell lysis often leads to stressed cells and higher amount of debris and dead cells, which bind unspecifically to the Microbeads.

You can recognize our engineered cytokines by the acronym “IS” in the product name. IS stands for improved sequence. Currently we offer human IL-2, FGF-2, Oncostatin M, PDGF-BB, VEGF, and mouse IL-3 as variants with an improved sequence. These engineered variants guarantee high stability, better solubility during reconstitution and high activity in culture.

MACS Cytokines are provided as lyophilized (freeze-dried) products. It is recommended to reconstitute lyophilized cytokines with deionized sterile-filtered water to a final concentration of 0.1–1.0 mg/mL in a minimal volume of 100 μL. Further dilutions should be prepared with 0.1% bovine serum albumin (BSA) or human serum albumin (HSA) in phosphate-buffered saline.

Upon reconstitution, store working aliquots at –20 °C or below, and avoid repeated freeze-thaw cycles. Shipment or short term storage f the lyophilized products at room temperature will not alter the performance.

The recovery of cells is often determined by comparing the number of cells isolated with the MACS Cell Separation Technology and the number of cells expected from the heterogeneous starting population.

However, for a more precise calculation, you count the cells in the original fraction, the cells in the target fraction, and the cells in the non-target fraction. The original fraction sample should be taken after magnetic labeling and directly before magnetic separation, as staining and centrifugation steps may lead to cell loss.

Cell recovery can be calculated as follows:

Target cell recovery

Recovery [%] = 100 x ([No. of cells in target fraction] x [% target cells in target fraction]) / ([No. of cells in original fraction] x [% of target cells in original fraction])

Overall cell recovery

Recovery [%] = 100 x ([No. of cells in target fraction + No. of cells in non-target fraction + No. of cells in wash fraction]) / [No. of cells in original fraction]

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).