Introduction

This product is intended for positive magnetic isolation of CD8+ T cells from lymphoid organs. The supplied protocol describes magnetic labeling and isolation from 5x107 cells. In the first step FlowComp™ Mouse CD8 Antibody (rat IgG2a antibody against mouse CD8) bind to the target cells. In the second step CD8+ T cells that have bound the specific antibodies are captured by the Dynabeads. In the last step beads are removed from the cells.

Downstream Applications

Isolated cells may be used directly in any downstream application such as flow cytometry. See recommended products and protocols.

Additional requirements

  • Isolation buffer: Ca2+ and Mg2+ free phosphate buffered saline (PBS) from Gibco (cat.no. 14190-094) supplemented with 0.1% BSA and 2mM EDTA (see Technical Support for further information).
  • Mixer allowing both tilting and rotation.
  • Magnet (DynaMag™ or Dynal MPC™)
  • Recommended Flow cytometry antibody reagents CD3-Alexa Flour 488 (Cat. No. HM3420);  CD8a-PE (Cat.No. MCD0804).
  • Recommendations for evaluation of viability is SYTOX Red (Cat. No. S34859).


Critical notes

  • Use a mixer that provides tilting and rotation of the tubes to ensure that Dynabeads do not settle at the bottom of the tube.
  • This product should not be used with magnet Dynal MPC™-1.
  • Never use less than recommended volume of Dynabeads.
  • Carefully follow the recommended pipetting volumes and incubation times.
  • Avoid air bubbles during pipetting.
  • For flow staining of cells after isolation it is recommended to use Caltag clone 5H10 as primary fluorescent antibody. Avoid using secondary antibodies specific for rat antibodies for flow cytometry staining.
  • Do not combine this kit with your own biotinylated antibody.

Protocol

Approximately 30–35% of mouse spleen cells are T cells, and about 30% of these T cells strongly express the CD8 antigen. This kit isolates highly pure CD8+ T cells. This protocol describes magnetic labeling and isolation o  CD8+ T cells from 5x107 mouse lymphoid cells using Dynabeads® FlowComp™ Mouse CD8.

Preparations

  • Prepare a single cell suspension from lymphoid organs (e.g. lymph nodes or spleen).
  • Prepare approximately 10 ml of isolation buffer per 5x107 cells.

Isolation procedure

When working with fewer cells than 5x107, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly.

  • Resuspend 5x107 cells in 500 μl isolation buffer and add 25 μl FlowComp™ Mouse CD8 Antibody.
  • ***  Mix well and incubate for 10 min at 2 – 8°C.
  • Add 2 ml cold isolation buffer to wash cells, followed by centrifugation for 8 min. at 350xg.
  • Remove and discard the supernatant.
  • Add 1 ml cold isolation buffer to the cell pellet and resuspend.
  • Add 75 μl resuspended FlowComp™ Dynabeads® and mix well.
  • ***   Incubate for 15 min at 2 – 8°C under rolling and tilting.
  • Place the tube in the magnet for minimum 1 min. Carefully remove and discard the supernatant.
  • Remove the tube from the magnet. Add at least 1 ml cold isolation buffer and resuspend the bead-bound cells by gentle pipetting 5 times.
  • Place the tube in the magnet for minimum 1 min. Carefully remove and discard the supernatant.
  • Remove the tube from the magnet and carefully resuspend the beadbound cells in 1 ml FlowComp™ Release Buffer.
  • ***   Incubate for 10 min. in room temperature under rolling and tilting.
  • Mix the cells by gentle pipetting 5 times and place the tube in the magnet for 1 min.
  • Transfer the supernatant containing the bead-free cells to a new tube and place the tube in the magnet for 1 min to remove all residual beads.
  • Transfer the supernatant containing the bead-free cells to a new tube.
  • Add 2 ml isolation buffer followed by centrifugation for 8 min. at 350xg.
  • 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.

***Incubation time


Further technical advice. For flow staining of cells after isolation it is recommended to use Caltag clone 5H10 as primary fluorescent antibody.

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114.62D.indd     Rev 003   10-Aug-2008