Introduction

DETACHaBEAD CD19 is intended for detachment of Dynabeads from CD19+ cells after isolation with Dynabeads CD19 Pan B, cat. no. 111.43D (not supplied). The detached CD19+ cells are pure, viable, not activated by the detachment and have no Dynabeads with primary antibody bound to the surface.

Principle of Detachment

DETACHaBEAD CD19 is added to the bead-bound cells after isolation with Dynabeads CD19 Pan B. The DETACHa-BEAD CD19 reagent will detach the Dynabeads and primary antibody from the CD19+ cells. Dynabeads are subsequently removed using a Dynal magnet, and the detached CD19+ cells are ready for any downstream application.

Description of Materials

DETACHaBEAD CD19 contains polyclonal anti-Fab antibodies specific for the CD19 antibody on the Dynabeads CD19 Pan B. No cross-reaction with human IgG, IgM or IgA has been detected in quantitative ELISA tests.

Materials Supplied

  • 5 ml DETACHaBEAD CD19 in 0.15 M phosphate buffered saline (PBS), pH 7.4.


Note:
Due to the high protein concentration it is not unusual to see some precipitation in the DETACHaBEAD CD19 component. This will not influence on product performance or quality.

Additional Materials Required

Materials that are not included, but are needed to perform the entire protocol:

  • Dynabeads CD19 Pan B (cat. no. 111.43D) for positive isolation of CD19+ cells.
  • Magnet: See www.lifetechnologies.com/magnets-selection for magnet recommendations.
  • Mixer allowing both tilting and rotation.
  • Tubes: We recommend adapting the tube size to the working volume (e.g. using 1.5 ml Eppendorf tubes for small volumes).
  • Medium, e.g. RPMI 1640 w/1% Foetal Bovine Serum (FBS).

Ordering Information

Sku Name Size Price Qty
12506D DETACHaBEAD® CD19 Kit 5 mL USD 550.00

Protocol

The following protocol is recommended for a starting sample of 2.5x107 MNC in 1 ml. The protocol is scalable, but a minimum detach volume (medium) and DETACHaBEAD is required (see below).

Table 1: Volumes of Dynabeads CD19 Pan B, medium and DETACHaBEAD CD19.

  Volumes
Volume Dynabeads CD19
Pan B used for cell isolation
25 μl
 Volume medium to resuspend
bead-bounds cells (step 2)
250 μl
Volume DETACHaBEAD CD19
(step 3)
10 μl

  1. Start with bead-bound cells after isolation with Dynabeads CD19 Pan B as recommended (i.e. use 25 μl of Dynabeads CD19 Pan B).

  2. Resuspend the bead-bound cells in 250 μl medium (never use less than 100 μl medium).

  3. Add 10 μl DETACHaBEAD CD19 per 25 μl Dynabeads used for cell isolation (never use less than 10 μl DETACHaBEAD CD19).

  4. Incubate for 45 min at room temperature with gentle mixing.

  5. Note: To avoid cell loss when working with small volumes, we recommend to keep the suspension in the bottom of the tube while still providing good mixing

  6. To enhance the release of cells, pipette the solution a few times before placing the tube in the magnet for approx. 1 min.

  7. Transfer the supernatant containing the released cells to a new tube. To obtain residual cells, wash the beads 2-3 times in 500 μl medium and pool the supernatants.

  8. Wash the detached cells thoroughly by resuspending the cells in a total volume of 10 ml medium and centrifuge for 6 min at 400 x g to remove any residual DETACHaBEAD reagent.

  9. Resuspend the cells in medium and use in any downstream application. The isolated CD19+ cells are pure  viable and are free from antibody or beads bound to the surface.
TOP

General Information

Invitrogen Dynal AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.

Storage/Stability

This product is stable until the expiry date stated on the label when stored unopened at 2-8°C. DO NOT FREEZE. Store opened vials at 2-8°C and avoid bacterial contamination.

Warnings And Limitations

This product is for research use only. Not for human or animal therapeutic or diagnostic use. Certificate of Analysis (CoA) is available upon request. Material Safety Data Sheet (MSDS) is available at

TOP

References

  1. Rasmussen AM, Smeland E, Erikstein BK, Caignault L and Funderud S. A new method for detachment of Dynabeads from positively selected B lymphocytes. J Immunol Methods 1992;146:195-202.

  2. Dryer RL and Covey LR (2006) Use of chromatin immuniprecipitation (ChlP) to detect transcription factor binding to highly homologous promoters in chromatin isolated from unstimulated and activated primary human B cells. Biol. Proced. Online;8(1):44-54.

  3. Døsen G et al (2006) Wnt expression and canonical Wnt signalling in human bone marrow B lymphopoiesis. BMC Immunology. www.biomedcentral.com/1471-2172/7/13.

  4. Ertesvag Aa et al (2007) Vitamin A potentiates CpG-mediated memory- B-cell proliferation and differentiation:

  5. involvement of early activation of p38MAPK. Blood 109(9):2865- 3872.

  6. Feederle R et al (2006) Epstein-Barr Virus BNRF1 Protein Allows Efficient Transfer from the Endosomal

  7. Compartment to the Nucleus of Primary B Lymphocytes. J. Virol. 80(19):9435-9443.

  8. Ryan EP et al (2005) Activated Human B Lymphocytes Express Cyclooxygenase-2 and Cyclooxygenase

  9. Inhibitors Attenuate Antibody Production. J Immunol. 174:2619-2626.
TOP
125.06D.indd   Rev 003   5-May-2007