BackgroundIsolate or deplete human CD4+ T cells directly from whole blood, buffy coat or MNC with Mouse anti-Human CD4 Particles. For rapid and consistent results in protein or gene expression analysis, lyse the CD4+T cells while they are still attached to the beads and directly process for further molecular analysis. The beads are mixed with the cell sample in a tube. Mouse anti-Human CD4 Particles bind to the target cells during a short incubation, and then the bead-bound cells are separated by a magnet Depletion – Discard the bead-bound cells and use the remaining, untouched cells for any application. Positive isolation – Discard the supernatant and use the bead-bound cells for downstream molecular applications.
Product Information
Experimental Methods1.Sample preparation: when working with anticoagulated peripheral blood should be isolated by density gradient centrifugation, for example, using Ficoll -paque. Add 2ml Isolation Buffer wash three times. Prepare PBMC to 1 × 107cells /ml in Isolation Buffer.
2.Transfer 1 ml cells (1 × 107) to a tube and add 50μL re-suspended Mouse anti-Human CD4 Particles. Incubate for 30 min at room temperature. With gentle tilting and rotation.
3.Place the tube in a magnet for 3-5 min.
For depletion; transfer supernatant to a new tube for further use and discard the beads.
For positive isolation; while the tube is still in the magnet, carefully remove and discard the supernatant.
4.Remove the tube from the magnet and add 1 ml Isolation Buffer, pipet 2–3 times and place the tube in a magnet for 3-5 min. While the tube is still in the magnet, carefully remove and discard the supernatant.
5.Repeat step 4 at 4-5 times. Wash the bead-bound CD4+ T cells. This step is critical to obtain a high purity of isolated cells.
6.Resuspend the cell pellet in preferred cell medium.
7.Keep the cells on 2°C to 8°C until further use in downstream applications.
Notices1.Use a mixer that provides tilting and rotation of the tubes to ensure that beads do not settle in the tube.
2.Isolation Buffer: Ca2+ and Mg2+ free PBS supplemented with 0.5% BSA and 2 mM EDTA, 0.09%NaN3, pH 7.4.
3.For research use only, not for diagnostic or therapeutic use.