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.

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.

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.

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.

BSA should be the first choice as protein supplementation for all separations.
If FCS is used instead, it should be used at the lowest possible concentration (range: 0.5–2%), and it should be replaced as soon as possible with BSA following the separation.

Yes, this is possible because all our protocols are standardized. It is important that you use the correct concentration of each type of MACS MicroBeads. Therefore, you will need to reduce the amount of buffer you add to the cell suspension.

For example, if you want to deplete T and B cells from PBMCs using CD3 MicroBeads, human and CD19 MicroBeads, human, follow the instructions in the CD3 MicroBeads data sheet as a base. Changes in the protocol (2.2 Magnetic labeling) compared the original version are marked in bold:

3. Resuspend cell pellet in 80 μL of buffer per 10⁷ total cells.
Since you are going to add 20 µL of CD19 MicroBeads as well, reduce this amount by 20 µL to 60 µL.

4a. Add 20 µL of CD3 MicroBeads per 10⁷ total cells.

4b. Add 20 µL of CD19 MicroBeads as well.

5. Mix well and incubate for 15 min at 4−8 °C.

You can typically combine up to three different types of MicroBeads. Combining more than three types might lead to difficulties with steric hindrances, so in these cases, we recommend that you use an indirect approach comparable to our isolation kits.

The Anti-Cy5/Anti-Alexa Fluor 647 MicroBeads recognize Cy5, Alexa Fluor 647, as well as tandem-conjugates with Cy5 or Vio700, such as PE-Cy7 or PerCP-Vio700.

We offer the CytoStim, human and the T Cell Activation/ Expansion Kit, human for polyclonal activation of human T cells.

Antigen-specific stimulation for CMV positive donors can be achieved with the CMV pp65 - Recombinant Protein or the PepTivator - CMV pp65, a peptide pool for CMV pp65-specific T cell stimulation.

MACS Separation Buffer can be ordered as autoMACS Running Buffer – MACS Separation Buffer. This buffer is sterile-filtered and ready to use. It contains bovine serum albumin (BSA), ethylene diamine tetraacetic acid (EDTA), and 0.09% azide.

Yes, this is possible. We recommend that you first stain with the Viobility Fixable Dye as described in the data sheet step 2.2, prior to staining for other surface or intracellular markers.

Afterwards, proceed with FcR blocking and antibody staining, as described in the respective data sheets of these products.

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).

We recommend SEB (Staphylococcal Enterotoxin B) for human or mouse samples. Furthermore, CytoStim, human or CMV antigen (only possible with CMV-positive donors) can be used as a positive control for human samples. Mitogens like PHA or PMA/Ionomycin are not recommended for stimulation of the positive control because the resulting high frequencies of cytokine-secreting (e.g., IFNγ) cells do not allow drawing conclusions about the performance (e.g., sensitivity) of the CSA.

All media are extensively tested for their performance. This includes functionality tests using the corresponding cell type in an appropriate experimental setting. Osmolality, pH and endotoxin levels are specified for complete and basal media.

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.

We recommend to follow our detailed application protocol "Isolation and cultivation of oligodendrocytes from adult mouse or rat brain". 

The culture dishes for cultivation of neural cells are typically coated with 0.01% Poly-L-Lysine overnight at 37°C. The dishes are then washed three times with sterile water and dried before plating the neural cells. For adult astrocytes, we additionally recommend a laminin coating (10 µg/mLl).

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

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 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.

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.

 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.

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.

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.

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.