BD IMag™ Anti-Rat CD8 Magnetic Particles - DM

MAGNETIC LABELING PROTOCOL

1.  Prepare a single-cell suspension from the lymphoid tissue of interest according to standard laboratory procedures. Remove clumps of cells and/or debris by passing the suspension through a 70-mm nylon cell strainer.

2.  Dilute BD IMag™ Buffer (10X) (Cat. no. 552362) 1:10 with sterile distilled water or prepare 1X BD IMag™ buffer by supplementing Phosphate Buffered Saline with 0.5% BSA, 2 mM EDTA, and 0.09% sodium azide.  Store at 4˚C.

3.  Wash cells with an excess volume of 1X BD IMag™ buffer, and carefully aspirate all the supernatant.

4.  Vortex the BD IMag™ anti-rat CD8 Particles - DM thoroughly, and add 50 μl of particles for every 10 million total cells.

5.  MIX THOROUGHLY.  Refrigerate at 6°C to 12°C for 30 minutes.

6.  Bring the BD IMag-particle labeling volume up to 10 to 80 million cells/ml with 1X BD IMag™ buffer, and immediately place the tube on the BD IMagnet™.  Incubate at room temperature for 8-10 minutes.

7.  With the tube on the BD IMagnet™, carefully pipette off the supernatant.  This supernatant contains the negative fraction.

8.  Remove the tube from the BD IMagnet™, and add 1 ml of 1X BD IMag™ buffer.  Gently resuspend cells by pipetting up and down, and return the tube to the BD IMagnet™ for another 2 - 4 minutes.

9.  With the tube on the BD IMagnet™, carefully pipette off the supernatant and discard.

10.  Repeat Steps 8 and 9.

11.  After the final wash step, resuspend the positive fraction in an appropriate buffer or media, and proceed with desired downstream application(s).

NOTE:  The concentration of BD IMag™ anti-rat CD8 Particles - DM suggested in the protocol has been optimized for the purification of CD8-positive T lymphocytes from rat splenocytes.  When labeling target cell populations present at lower frequencies, fewer BD IMag™ particles can be used.  Conversely, when labeling target cell populations that are present at higher frequencies, more particles should be used.  To determine the optimal concentration of the BD IMag™ anti-rat CD8 Particles - DM for a particular application, a titration in two-fold increments is recommended.

NOTE:  Avoid nonspecific labeling by working quickly and keeping incubation times as recommended.

1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. BD IMag™ particles are prepared from carboxy-functionalized magnetic particles which are manufactured by Skold Technology and are licensed under US patent number 7,169,618.
3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
4. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.