磁珠分选淋巴细胞sop.pdfVIP

SOP for Magnetic Bead Separations (using “Positive Selection” of White Blood Cell Subsets from PBMCs By Daniel Estes, 2006 Modified: DJE, 2007 General Using magnetic beads that attach to CD8 or CD4 on T-cells, or CD19 on B cells, is an effective way to positively select for and isolate white blood cell subsets with 97% purity from PBMCs. The method employs a magnet to “capture” cells with the magnetic beads, and when the magnet is removed, the cells are released. Note that these instructions are similar to those provided by Miltenyi biotech except for three changes: 1) separations occur at room temperature to avoid any sort of temperature “shock” to cells; 2) we use half the recommended amount of bead solution; and 3) for all buffers, we use the simplified wash buffer below instead of a specific magnetic bead buffer. The buffer below provides a total separation time of 15 minutes (of actually dripping through the columns), is reliable (we never have any clogging of the columns). BE CAREFUL : PBMCs as well as isolated T cells may contain bloodborne pathogens, so always wear appropriate laboratory attire, dispose of cells in appropriate manner, and follow general blood protocols (please see Mayer lab Bloodwork SOP). Materials - Wash buffer (98% dPBS (w/o Ca2+ and Mg2+), 2% FBS) - MicroBeads coated with antibodies to either human CD4 or CD8 (Miltenyi Biotec) - MACS MultiStand - MS Columns - MiniMACS Magnet Procedure General notes: work quickly and wear appropriate protection attire (see bloodwork and cell culture SOPs). NOTE: if sterility is required, first wash all components of setup in 70% ethanol and do separations in laminar flow hood. 1. Starting with PBMCs that have been thoroughly washed (obtained in our case after Histopaque separation) 2. Determine cell count – please note that the amounts of bead solution indicated in step #4 are only good for a maximum of 50 million cells 3. Spin-down cells (I usually use 300g’s fo