Anti-R-Phycoerythrin Magnetic Particles

Produktnummer ABIN1305247

Produktdetails

Target: R-Phycoerythrin (Phycoerythrin, R-)

Reaktivität: Maus

Wirt: Maus

Klonalität: Monoklonal

Konjugat: Magnetic Particles

Applikation: Separation (Sep)

Marke: BD IMag™

Anwendungsinformationen

Protokoll: 1. Prepare the following buffers and place on ice. a. Cell-staining buffer: Phosphate Buffered Saline, 3% heat inactivated fetal calf serum, 0.1% sodium azide. b. 1X BD IMag™ buffer: Dilute BD IMag™ Buffer (10X) (Cat. no. 552362) 1:10 with sterile distilled water or alternatively, prepare Phosphate Buffered Saline, supplemented with 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide.
2. Prepare a single-cell suspension from the lymphoid tissue of interest or prepare PBMC from anti-coagulated blood, preferably by density gradient centrifugation using the appropriate density Ficoll-Hypaque™ solution. Remove clumps of cells and/or debris by passing the suspended cells through a 70-µm nylon cell strainer.
3. Count the cells, and resuspend them in cell-staining buffer at a concentration of 2 x 10e7 cells/ml.
4. Optional: If appropriate, add BD Mouse Fc Block™ purified anti-mouse CD16/CD32 mAb 2.4G2 (Cat. No. 553141) or BD Rat Fc Block™ purified anti-rat CD32 mAb D34-485 (Cat. No. 550270) at 0.25 µg/10e6 cells, and incubate on ice for 15 minutes.
5. Add the PE-conjugated antibody (or cocktail of PE-conjugated antibodies) at the appropriate concentration, and incubate on ice for 15 minutes.
6. Wash the labeled cells with an excess volume of 1X BD IMag™ buffer, and carefully aspirate ALL the supernatant. For depletions, proceed with Step 7. For positive selections, proceed with Step 18.

Depletions:
7. Vortex the BD IMag™ Anti- R-Phycoerythrin (PE) Magnetic Particles - DM thoroughly, and add 50 µl of particles for every 1 x 10e7 total cells.
8. MIX THOROUGHLY. Refrigerate mouse or rat leukocytes for 30 minutes at 6°C
- 12°C. Incubate human PBMC at room temperature for 30 minutes.
9. Bring the labeling volume up to 2-8 x 10e7 cells/ml with 1X BD IMag™ buffer or culture medium.
10. Transfer the labeled cells to a 12 x 75 mm round-bottom test tube (eg, BD Falcon™, Cat. No. 352058), maximum volume added not to exceed 1.0 ml. Place this positive-fraction tube on the BD IMagnet™ (horizontal position) for 6-8 minutes. - For greater volume, transfer the cells to a 17 x 100 mm round-bottom test tube (eg, BD Falcon™, Cat. No. 352057), maximum volume added not to exceed 3.0 ml. Place this positive-fraction tube on the BD IMagnet™ (vertical position) for 8 minutes.
11. With the tube on the BD IMagnet™ and using a glass Pasteur pipette, carefully aspirate the supernatant (depleted fraction) and place in a new tube.
12. Remove the positive-fraction tube from the BD IMagnet™, and add 1X BD IMag™ buffer (or medium) to the same volume as in Step 9. Resuspend the positive fraction well by pipetting up and down 10-15 times and place back on the BD IMagnet™ for 6-8 minutes.
- 17 x 100 mm tube: Place on the BD IMagnet™ for 8 minutes.
13. Using a new Pasteur pipette, carefully aspirate the supernatant and combine with the depleted fraction from Step 11 above.
14. Repeat Steps 12 and 13. The Combined Depleted Fraction contains cells with no bound antibodies or magnetic particles. These cells are ready for downstream applications, or they can be further enriched by proceeding to Step 16.
15. The positive-fraction cells remaining in the original tube can be resuspended in an appropriate buffer or culture medium for downstream applications, including flow cytometry.
16. To increase the purity of the Combined Depleted Fraction, place the tube on the BD IMagnet™ for another 6-8 minutes. - 17 x 100 mm tube: Place on the BD IMagnet™ for 8 minutes.
17. Carefully aspirate the supernatant and place in a new tube. This is the Final Depleted Fraction. The cells are ready to be processed for downstream applications.

Positive Selections:
18. Vortex the BD IMag™ Anti- R-Phycoerythrin (PE) Magnetic Particles - DM thoroughly, and add 10-50 µl of particles for every 1 x 10e7 total cells.
NOTE: The amount of particles to add may vary depending on how many cells one is targeting and the cell-surface density of the antigen. Please refer to Table 1 for some common examples.
19. MIX THOROUGHLY. Refrigerate mouse or rat leukocytes for 30 minutes at 6°C
- 12°C. Incubate human PBMC at room temperature for 30 minutes.
20. Bring the labeling volume up to 2-8 x 10e7 cells/ml with 1X BD IMag™ buffer.
21. Immediately place the tube onto the BD IMagnet™ and incubate for 6-8 minutes.
22. With the tube on the BD IMagnet™, carefully aspirate the supernatant. This supernatant is considered the Negative Fraction.
23. Remove the tube from the BD IMagnet™, and add 1X BD IMag™ buffer to the same volume as in Step 20. Gently resuspend the cells by pipetting up and down, and return the tube to the BD IMagnet™ for another 2-4 minues.
24. With the tube on the BD IMagnet™, carefully remove the supernatant.
25. Repeat Steps 23 and 24.
26. After the final wash step, remove the tube from the BD IMagnet™. Resuspend the Positive Fraction in an appropriate buffer or culture medium, and proceed with desired downstream application(s), including flow cytometry.

NOTES: For depletion of mouse leukocytes, tissue culture medium usually results in a slight increase in viability and recovery, when compared to IMag buffer, without reducing cell purity. Because applications can vary, researchers are encouraged to run a trial comparison of culture media and IMag buffer to demonstrate that there are no adverse effects
Avoid non-specific labeling by working quickly and adhering to recommended incubation times.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Format: Liquid

Buffer: Aqueous buffered solution containing BSA and ≤0.09% sodium azide.

Konservierungsmittel: Sodium azide

Vorsichtsmaßnahmen: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Lagerung: 4 °C

Informationen zur Lagerung: Store undiluted at 4° C. Table 1. Various reported optimal concentrations of BD IMag™ Anti- R-Phycoerythrin (PE) Magnetic Particles - DM for positive selection with some PE-conjugated monoclonal antibodies to human and mouse leukocyte antigens.

Antigendetails

Target: R-Phycoerythrin (Phycoerythrin, R-)

Andere Bezeichnung: R-Phycoerythrin (R-Phycoerythrin Produkte)

Substanzklasse: Chemical

Hintergrund: BD IMag™ Anti- R-Phycoerythrin (PE) Magnetic Particles - DM are magnetic nanoparticles that have monoclonal antibody conjugated to their surfaces. The E31-1459 antibody clone reacts with PE, a commonly used fluorochrome for flow cytometry. The binding of the E31-1459 antibody to PE has been reported not to quench the fluorescence of the PE molecule. These magnetic particles are optimized for the positive selection or depletion of leukocyte subpopulations using the BD IMagnet™. Leukocytes are labelled with BD IMag™ Anti- R-Phycoerythrin (PE) Magnetic Particles - DM according to the Magnetic Labeling and Separation Protocol. In brief, cells are labeled with a PE-conjugated antibody that recognizes the subpopulation of interest. After washing away excess antibody, BD IMag™ Anti- R-Phycoerythrin (PE) Magnetic Particles - DM are added to the cell suspension and bind the PE-conjugated antibody on the cells. This labeled cell suspension is then placed within the magnetic field of the BD IMagnet™.