Option 1: Automated Immunomagnetic Cell Isolation with EasySep™ Direct Human PBMC Isolation Kit

Materials

• Leukapheresis pack (e.g. Catalog #70500)*
• EasySep™ Direct Human PBMC Isolation Kit (Catalog #19654 or #19654RF)
• EasySep™ Magnet (e.g. Catalog #18000 for manual cell isolation or RoboSep™-S (Catalog #20001) for automated cell isolation
• Recommended medium (medium should be free of Ca++ and Mg++). Options include:
  • EasySep™ Buffer (Catalog #20144) or
  • RoboSep™ Buffer (Catalog #20104) or
  • D-PBS (Without Ca++ and Mg++; Catalog #37350) or
  • PBS containing 2% FBS and 1 mM EDTA
• Ethylenediaminetetraacetic acid (EDTA)
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• 14 mL polystyrene round-bottom tubes (e.g. Catalog #38008)
• 50 mL conical tubes (e.g. Catalog #38010)

*Certain products are only available in select territories. Please contact your local Sales representative or the Product & Scientific Support team at techsupport@stemcell.com for further information.

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Protocol

Below is an example of how to concentrate a full-size leukapheresis pack and then perform PBMC isolation using RoboSep™-S. For manual immunomagnetic cell isolation, follow the instructions in the Product Information Sheet - Leukapheresis for using EasySep™ Direct Human PBMC Isolation Kit.

1. Transfer the entire contents of the leukapheresis bag into tubes. Centrifuge at 300 x g for 10 minutes with the brake ON.
2. Carefully remove the supernatant and resuspend the cells in recommended medium to obtain a final volume of 20 mL and approximate concentration of 4 x 10⁸ cells/mL.
3. Add EDTA at 6 mM final concentration and aliquot the 20 mL into 4 x 14 mL polystyrene round-bottom tubes. Each tube will now contain 5 mL of resuspended cells.
4. Select the appropriate protocol on the instrument’s screen.
5. Follow the onscreen prompts and load the 4 x 14 mL tubes, reagents, tips, and buffer onto the RoboSep™-S carousel.
6. Press “Run”.
7. Come back in 30 minutes after the run is complete and collect the isolated cells.

Option 2: RBC Lysis with Ammonium Chloride Lysis

Materials

• Leukapheresis pack (e.g. Catalog #70500)*
• Ammonium Chloride Solution (Catalog #07800)
• 50 mL conical tubes (e.g. Catalog #38010)
• Recommended medium. Options include:
  • EasySep™ Buffer (Catalog #20144) or
  • PBS containing 2% FBS (e.g. Dulbecco's Phosphate Buffered Saline with 2% Fetal Bovine Serum, Catalog #07905)
• Sterile bottle

Protocol

1. Transfer the contents of the leukapheresis pack into a sterile bottle. Please refer to Part I of our Leukapheresis Processing Protocol for more information on handling leukapheresis samples.
2. Add Ammonium Chloride Solution to the sample at a 1:4 ratio (e.g. 50 mL sample + 200 mL Ammonium Chloride Solution). Mix thoroughly.
3. Incubate on ice for 15 minutes.
4. Transfer to 50 mL conical tubes. Centrifuge at 500 x g for 10 minutes at room temperature (15 - 25°C) with the brake ON.
5. Remove the supernatant. Gently resuspend the cell pellet, and top up to 50 mL with recommended medium. Centrifuge at 300 x g for 10 minutes at room temperature (15 - 25°C) with the brake ON.
6. Repeat wash step 5.
   
   

   Optional: If desired, perform a platelet removal wash instead of a regular cell wash by centrifuging at 120 x g for 10 minutes at room temperature (15 - 25°C) with the brake OFF. Additional platelet washes may be required depending on donor variability.

7. Remove the supernatant and resuspend the PBMC pellet in appropriate medium.

Option 3: Density Gradient Centrifugation

Materials

• Leukapheresis pack (e.g. Catalog #70500)*
• Density gradient medium (e.g. Lymphoprep™, Catalog #07851)
• 50 mL conical tubes (e.g. Catalog #38010)
• Recommended medium. Options include:
  • EasySep™ Buffer (Catalog #20144) or
  • PBS containing 2% FBS (e.g. Dulbecco's Phosphate Buffered Saline with 2% Fetal Bovine Serum, Catalog #07905)
• Sterile bottle
• Serological pipettes (e.g. Falcon® Serological Pipettes, 25 mL, Catalog #38005)

Protocol

Before You Begin: Ensure all samples and reagents are at room temperature (15 - 25°C) prior to starting this protocol.

1. Transfer the contents of the leukapheresis pack into a sterile bottle. Refer to Part I of our Leukapheresis Processing Protocol for more information on handling leukapheresis samples.
2. Dilute the leukapheresis sample 1:1 with recommended medium (e.g. 50 mL sample + 50 mL EasySep™ Buffer or PBS containing 2% FBS). Wash the leukapheresis bag with the buffer used for dilution.
   
   

   Note: In some cases where the leukocyte concentration is high, a 1:2 dilution (e.g. 50 mL sample + 100 mL medium) can help improve separation.

3. Add 15 mL of density gradient medium to a 50 mL conical tube.
4. Slowly layer 30 mL of diluted leukapheresis sample over the density gradient medium.
5. Centrifuge at 800 x g for 20 minutes at room temperature (15 - 25°C) with the brake off.
   
   

   Note: If the blood has been stored for more than 2 hours, increase the centrifugation time to 30 minutes.

6. Remove and discard the upper plasma layer without disturbing the plasma:density gradient medium interface.
7. Collect the PBMC layer at the plasma:density gradient medium interface without disturbing the RBC/granulocyte pellet.
8. Wash the isolated PBMCs with recommended medium. Centrifuge at 300 x g for 10 minutes at room temperature (15 - 25°C) with the brake ON.
   
   

   Optional: If desired, perform a platelet removal wash instead of a regular cell wash by centrifuging at 120 x g for 10 minutes at room temperature (15 - 25°C) with the brake OFF. Additional platelet washes may be required depending on donor variability.

9. Remove the supernatant and resuspend the PBMC pellet in appropriate medium.