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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.
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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 10 7 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 10 7, 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