Part I: Transfer LRS Cone Contents to a Sterile Container

1. First, carefully clean the external surface of the LRS cone and the tubing with 70% isopropyl alcohol or ethanol.
2. Then place the LRS cone, wide side down, over a 50 mL conical tube. This is the Collection Tube.
3. With the LRS cone held over the Collection Tube, use sterile scissors to cut the bottom tubing located at the wider side of the cone, leaving approximately 2 to 3 cm of tubing.
4. 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.
5. To facilitate the transfer of the sample into the Collection Tube, an air flush can be performed. To do this:
   1. Use a pipette to insert a sterile 200 µL tip into the tubing at the top of the LRS cone.
   2. Gently twist the tip several times to ensure it is tightly positioned before releasing the tip inside the tubing.
   3. 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.
   4. Continue to apply air until the sample, typically 8 to 10 mL, has completely transferred into the Collection Tube.
6. 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.
7. Then perform an air flush.
8. 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. After the final air flush, the sides of the cone should look clear. The cone can then be discarded.
   Note: Ensure that the collected wash solution does not contact the tubing at the bottom of the cone.
9. Top up the diluted sample to 50 mL with PBS containing 2% FBS.
10. Cap the tube securely, and gently mix the diluted sample by inverting the tube 5 to 6 times.
11. Centrifuge the tube at 800 x g for 10 minutes with the brake on or with deceleration set to high.
12. Using a 10 mL serological pipette, carefully remove the supernatant without disturbing the LRS cone pellet. Leave some supernatant to maintain a total volume equal to the original sample (approximately 8–10 mL).
13. Resuspend the LRS cone pellet by gently flicking or shaking the tube back and forth until the cells are thoroughly dispersed.

Part II: Prepare LRS Cone Contents

All three of the following protocol options are effective for preparing nucleated cells from LRS cone/chamber samples for subsequent cell isolation. The protocol should be chosen based on the intended target cells, cost-effectiveness,and processing time constraints.

LRS cone samples typically contain a low frequency of granulocytes; both density gradient centrifugation (Option 2) and the EasySep™ Direct Human PBMC Isolation Kit (Catalog #19654) protocol (Option 3) deplete granulocytes and yield PBMCs with high purity. Conversely, the RBC lysis method (Option 1) does not achieve granulocyte depletion, but is a more cost-effective and faster method, providing sufficient PBMC purity for further cell isolation applications. For further guidance on the optimal method for your specific application, please contact us at techsupport@stemcell.com.

Option 3: EasySep™ Direct Human PBMC Isolation Kit (Catalog #19654)

PBMCs can also be easily isolated directly from blood without centrifugation or lysis using the EasySep™ Direct Human PBMC Isolation Kit. This simple and fast immunomagnetic isolation method results in purified PBMCs from an LRS cone. Consult the leukoreduction system chamber-specific Product Information Sheet for detailed instructions on isolating PBMCs.