The protocol below outlines how to process a leukocyte reduction system (LRS) cone for isolating cells using manual or automated immunomagnetic and column-free EasySep™ cell isolation kits. Following immunomagnetic cell separation, the untouched cells are immediately ready for downstream analysis.
Serological Pipettes (25mL- e.g. Catalog #38005, 10mL- e.g. Catalog #38004)
Before You Begin: Ensure that you handle the LRS cone and its contents aseptically within a biosafety cabinet.
First, carefully clean the external surface of the LRS cone and the tubing with 70% isopropyl alcohol or ethanol.
Then place the LRS cone, wide side down, over a 50 mL conical tube. This is the Collection Tube.
With the LRS cone held over the Collection Tube, cut the bottom tubing located at the wider side of the cone with sterile scissors, leaving approximately 2 to 3 cm of tubing.
Return the LRS cone to rest on the Collection Tube. Then cut the top tubing, located at the narrower side of the cone, leaving approximately 1 cm of tubing. The sample will now start to drip into the Collection Tube.
To facilitate the transfer of the sample into the Collection Tube, an air flush can be performed. To do this:
a) use a pipette to insert a sterile 200 µL tip into the tubing at the top of the LRS cone. b) Gently twist the tip several times to ensure it is tightly positioned before releasing the tip inside the tubing. c) Attach a blunt-end 16-gauge needle securely to a 12 mL syringe, and use the syringe to push air into the 200 µL tip. d) Continue to apply air until the sample, typically 8 to 10 mL, has completely transferred into the Collection Tube.
Next, to rinse the cone, fill the syringe with a buffer solution of PBS containing 2% FBS. Gently dispense the buffer solution through the 200 µL tip at a rate of approximately 2 mL/second. Be sure to move the syringe in a circular motion to help wash the sample from the sides of the cone.
Then perform an air flush.
Repeat the wash and air flush steps (steps 6 and 7) two additional times, until the cone has been rinsed with approximately 30 mL of buffer solution.
Note: Ensure that the collected wash solution does not contact the tubing at the bottom of the cone.
Note: After the final air flush, the sides of the cone should look clear. The cone can then be discarded.
Top up the diluted sample to 50 mL with PBS containing 2% FBS.
Cap the tube securely, and gently mix the diluted sample by inverting the tube 5 to 6 times.
Centrifuge the tube at 800 x g for 10 minutes with the brake on or with deceleration set to high.
After spinning, use a 10 mL serological pipette to carefully remove the supernatant.
Note: Be careful not to disrupt the bottom layer containing the leukocytes and the red blood cells.
Mix the sample (~ 10mL) by gently flicking or shaking the tube back and forth. The cells are now ready for downstream applications, but may require additional processing steps for some EasySep™ cell separation protocols.
Note: If not used immediately, the cells can be stored at 2 - 8°C for up to 48 hours.
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Processing Leukocyte Reduction System (LRS) Cones/Chambers for Downstream Cell Isolation