Below are the key points to achieve good result in cell separation procedures using MACS.
- Prepare separation buffer by following the datasheet.
- Degas separation buffer before use!!! This is very important to prevent column clogging caused by air bubbles. Degassed buffer can be prepared by filtering buffer using 0.22 μm filter. Keep the buffer cold and use it within 72 hrs. Beyond that, buffer needs to be re-degassed.
- Always start with fresh samples as much as possible. If initial sample source is tissue sample and it cannot be processed immediately, MACS Tissue Storage Solution will be an option to maintain the good condition of the sample.
- Prepare single-cell suspension and filter cell suspension through 30 μm Pre-Separation Filter, in order to remove cell clumps.
- Count cell number (original fraction) and adjust column number accordingly. There is a limitation for the total cell number and Microbeads labeled cell number. Do not overload column and exceed the column capacity. Refer to the datasheet for column capacity for different cell types.
- Check cell viability by using live/dead dyes, e.g. trypan blue and propidium iodide. Dead cells may bind non-specifically to MACS MicroBeads. To remove dead cells, use density gradient centrifugation or the Dead Cell Removal Kit.
Magnetic Cell Labeling
- The three most important parameters are time, temperature and MicroBeads concentration. Follow the protocol strictly.
- Higher temperatures or longer incubation times lead to non-specific cell labeling. Incubation on ice requires increased incubation time, otherwise cells might be insufficiently labeled.
- The incubation time for magnetic labeling is 15 minutes for most MACS cell separation reagents, preferably at 4–8 °C. Refer to the datasheet for optimal incubation times/temperature.
Magnetic Cell Separation
- Aliquot small portion of the labeled cells and keep them on ice as original fraction for quality control.
- Load cells on the column and perform cell separation according to the datasheet.
- To increase the purity of the target cells, collected cells can be passed over a new, freshly prepared MS Column or LS Column.
- Collect positive cells (with MicroBeads labeled) and negative cells (without MicroBeads labeled). Count cell number and check viability with live/dead dye. Aliquot a small portion of each cell fraction (positive and negative) for quality control.
- Check cell purity by flow analysis: stain aliquoted cell fractions (original, positive and negative) with cell specific markers. Analyze cells with flow cytometer to determine purity/frequency of target cells before and after separation.
- Separation recovery = [Cell count of target cells fraction × purity of target cells (post separation)] / [Cell count of original fraction × frequency of target cell in original fraction (before separation)] × 100%