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| Questions & answers |
| Can a certain concentration of antibody be recommended for labeling of cells with a primary antibody? |
| There is no defined antibody concentration that can generally be recommended for labeling of cells with a primary antibody. Each suitable antibody concentration depends on the target antigen expression on the cell surface, as well as on the affinity of the individual antibody. Therefore, the ideal concentration of a primary antibody has to be determined experimentally. |
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| How can the ideal concentration of an antibody for primary labeling be determined? |
Different concentrations of a fluorochrome-conjugated primary antibody are used for primary labeling. The following recommendations can be used as a guideline:
- Perform a log2 dilution series starting with 2 µg/100 µL, 1 µg/100 µL, 0.5 µg/100 µL down to 0.016 µg/100 µL.
- Suspend 106 cells in 50 µL of buffer and add 50 µL of the antibody dilutions.
- Incubate for 10 min at 4–6 °C.
If an unconjugated or biotinylated antibody is chosen for primary labeling, an additional staining step has to be applied before flow cytometric analysis. The second labeling step is performed with a fluorochrome-conjugated antibody directed against the primary antibody, e.g., Anti-IgG-FITC in case of a primary IgG-isotype antibody, or Anti-Biotin-PE in case of a biotinylated primary antibody. Of course, the fluorochrome-staining reagent itself has to be applied at its ideal concentration.
The cells are then washed, suspended in flow buffer, and analyzed by flow cytometry. |
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| Why is it important to wash the cells after incubation with a primary antibody? |
| The washing step is necessary to remove free, unbound primary antibody. If this washing step is not performed, free primary antibody, not bound by the target cell, may interfere with the following magnetic labeling with MACS® MicroBeads. This may lead to a decreased magnetic labeling of target cells and can finally result in a low recovery of positively selected cells. |
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| What happens if a suboptimal concentration of a primary antibody is used? |
The ideal concentration of primary antibody leads to bright labeling of target cells combined with the least possible non-specific staining of non target cells, measurable by flow cytometry.
- Too high concentrations of primary antibody may lead to unspecific labeling of non-target cells. In such a case, target cells as well as non-target cells will be magnetically labeled during the incubation with MACS® MicroBeads for indirect magnetic labeling. This will reduce purity of the selected positive fraction. In case of a depletion strategy, it may lead to a lower recovery of the unlabeled target cells. In both cases, a certain amount of the undesired unspecifically magnetically labeled cells will be retained on the column.
- Too low concentration of primary antibody can lead to an insufficient magnetic labeling of target cells, resulting in low recovery of the positive fraction (positive selection) or in reduced purity of the negative fraction (depletion).
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