Introduction

This product is intended for positive magnetic isolation of CD49b+ NK cells from mouse splenocytes. The supplied protocol describes magnetic labeling and isolation from 5 x 107 spleen cells. In the first step, FlowComp™ Mouse CD49b antibody binds the target cells. In the second step, the labeled CD49b+ NK target cells are captured by the Dynabeads®. In the last step, the beads are removed from the cells.

Downstream applications

Isolated cells are bead-free and may be directly analyzed by flow cytometry and used in any downstream application such as expansion or cytotoxicity assays (i.e. Crrelease and LAMP-1 expression).

Additional materials required

  • Isolation buffer: Ca2+ and Mg2+ free phosphate buffered saline (PBS) (e.g. Gibco cat.no. 10010-023) supplemented with 0.1% BSA and 2mM EDTA.
  • Mixer allowing both tilting and rotation.
  • Magnet (Dynal Mag™ or Dynal MPC™): See magnet recommendations.
  • Flow cytometry antibody reagents (optional): We recommend using anti-CD49b clone Ha1/29.


Note:

  • BSA can be replaced by human serum albumin (HSA) or 2% FBS/FCS.
  • EDTA can be replaced by 0.6% sodium citrate.


Critical notes:

  • Wash the FlowComp™ Dynabeads® prior to use (critical for recovery).
  • Use a mixer that provides tilting and rotation of the tubes to ensure that Dynabeads do not settle in the tube.
  • This product should not be used with Dynal MPC™-1 magnet (Cat.no. 120.01D).
  • Avoid air bubbles during pipetting.
  • Never use less than recommended volume of Dynabeads®.
  • Carefully follow the recommended pipetting volumes and incubation times.
  • Avoid using secondary antibodies specific for mouse antibodies when multi-staining is performed.
  • Do not combine this kit with your own biotinylated antibody, since the cells will not be released from the FlowComp™ Dynabeads®.

Protocol

Approximately 6-8% of mouse spleen cells express CD49b, including most subsets of NK cells, subsets of NKT cells, γδ TCR T cells and some myeloid cells. CD49b is expressed by most commonly used inbred mouse strains, such as BALB/c and C57Bl/6. This protocol describes magnetic labeling and isolation of highly pure CD49b+ NK cells from 5 x 107 mouse splenocytes using Dynabeads® FlowComp™ Mouse CD49b.

Dynabeads® Washing Procedure

  1. Resuspend the Dynabeads® in the vial.

  2. Transfer the desired volume of Dynabeads® to a tube.

  3. Add the same volume of isolation buffer, or at least 1 ml, and mix.

  4. Place the tube in a magnet for 2 min and discard the supernatant.

  5. Remove the tube from the magnet and resuspend the washed Dynabeads® in the same volume of isolation buffer as the initial volume of Dynabeads® (step 2).

Preparations

  • Prepare mouse splenocyte single cell suspension from mouse spleens using standard procedure (see Technical Support for further information).
  • Prepare a single cell suspension of 1 x 108 cells/ml in isolation buffer.
  • Prepare approximately 10 ml isolation buffer per 5 x 107 cells.

Isolation procedure

When working with fewer cells than 5 x 107, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent and total volumes accordingly.

  1. Resuspend 5 x 107 cells in 500 μl (1 x 108 cells/ml) isolation buffer and add 25 μl FlowComp™ Mouse CD49b Antibody.

  2. Mix well and incubate for 15 min at 2–8°C.

  3. Add 2 ml isolation buffer to wash cells, followed by centrifugation for 8 min at 350xg.

  4. Remove and discard the supernatant.

  5. Add 0.5 ml isolation buffer to the cell pellet and resuspend.

  6. Add 75 μl pre-washed and resuspended FlowComp™ Dynabeads® (mCD49b) and mix well.

  7. Incubate for 15 min at 2–8°C under rolling and tilting.

  8. Place the tube in the magnet for minimum 1 min. Carefully remove and discard the supernatant.

  9. Remove the tube from the magnet. Add at least 2 ml isolation buffer and resuspend the bead-bound cells by gently pipetting 5 times.

  10. Place the tube in the magnet for minimum 1 min. Carefully remove and discard the supernatant.

  11. Remove the tube from the magnet and carefully resuspend the beadbound cells in 1 ml FlowComp™ Releas  Buffer.

  12. Incubate for 20 min at room temperature under rolling and tilting.

  13. Mix the cells by pipetting 10 times and place the tube in the magnet for 1 min.

  14. Transfer the supernatant containing the bead-free cells to a new tube.

  15. Add 2 ml Isolation Buffer followed by centrifugation for 8 min at 350xg.

  16. Discard the supernatant and resuspend the cell pellet in preferred cell medium.

Keep the cells on 2–8°C until further use in downstream applications. Learn more

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114.64D.indd     Rev 003   10-Aug-2008