Institute of Life Sciences (ILS) and Abgenex are proud to introduce QuikSort™, a magnetic nanoparticles based system for effective cell isolation. Cells are isolated through positive selection, where cells of interest are bound by antibody-conjugated magnetic nanobeads. The isolated CD3 cells can be used for specific research purpose, such as downstream applications include functional assays, gene expression, phenotypic characterization, etc.
Kit Contents
Supplied Material
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Magnetic device (Cat No. 25-1000)
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Buffer A (Binding buffer, Cat No. 2001 A)
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Buffer B (Elution buffer, Cat No. 2001 B)
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Vials containing lyophilized anti-CD3 labeled magnetic nanoparticles vial is meant for two reactions.
Instruments and Reagents Required
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Rotary shaker
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Vortex
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5 ml (12 mm x 75 mm) polystyrene tube
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Fluorescence labeled anti-mouse CD3 antibody and Flow Cytometer.
Note: Re-suspend each lyophilized vial in binding buffer (Cat No. 25001 A)
Detailed protocol and flow chart are included in the KIT as separate sheet.
This kit is designed for the isolation of CD 3 cells from splenocytes.
Protocol
Step 1. Take 5 X 106 mouse splenocytes in 1ml 1X Buffer A in a polystyrene tube, to that add 1 ml anti-CD3 labeled
MNPs suspension. The cell and the buffer mixture volume should be 2 ml, thereafter seal the tube with parafilm. Keep it in rotary shaker at 4°C for 30 minutes for binding of magnetic beads to the cells. After the incubation period, take out the tube containing the cell suspension and place the tube in the magnetic device (as shown in the picture). Gently pipette out the supernatant while keeping the tube inside the magnetic device. The obtained pellet contains CD3+ cells, bound to magnetic beads.
Step 2. Keep the above pellet in the magnetic device and add 1 ml 1X Buffer A. Pipette out the supernatant while keeping the tube inside the magnetic device.
Step 3. Remove the tube containing the pellet from the magnetic device, to that add 800 µll chilled Buffer B and gently mix the cell suspension thrice by pipette, keep in ice for 90 seconds. After the incubation gently mix the cell suspension by pipette and proceed to step no 4.
Note: Keep the Buffer B in chilled condition throughout the experiment.
Step 4. Place the tube containing cell suspension in the magnetic device. Collect the supernatant containing the desired CD3 positive cells into another 2 ml of microfuge tube containing 200 µl of Buffer A. Repeat the above process twice by adding 500 µl of Buffer B. Pool all the eluted cells in above microfuge tube and centrifuge at 2000 rpm for 10 min at 4°C.
Step 5. Thereafter, remove the supernatant carefully, leaving about 100 µl of Buffer containing the purified CD3+ cells.
Step 6. Add 200 µl of 1X Buffer A into the cell suspension and stain the cells with fluorescence labeled anti-mouse CD3 antibody keep in ice for 30-45 mins. After that add 500 µl of buffer A, centrifuge the cells at 2000 rpm for 10 min at 4°C. Remove 300 µl of supernatant by keeping 200 µl of buffer containing desired cell suspension.
Step 7. Add 400 µl of buffer A and mix gently, and analyze by Flow Cytometry.
Note: The procedure is optimized for 5 X 106 cells
