Application Note

Washing MAGPIX beads with the AquaMax 2000/4000 Microplate Washer and LifeSep magnetic plate holder

  • Optimized washing for high-throughput bead-based multiplexing
  • Excellent retention of washed beads
  • Highly adjustable wash settings for maximum flexibility

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Introduction

The MAGPIX multiplexing system offers high-throughput multiplexing capabilities with up to 4800 results in one hour. To ensure optimal performance of these assays, it is essential to wash the MAGPIX beads efficiently while maximizing retention of the beads within the microplate wells. With the AquaMax® 2000/4000 Microplate Washers from Molecular Devices®, bead retention of at least 95% is achievable using the optimized settings described in this document.

magapix-beads-one

Figure 1. AquaMax 4000 Microplate Washer using the MAGPIX multiplexing system and a LifeSep magnetic plate holder.

Materials

  • MAGPIX Calibration Kit (Life Technologies, Cat. #MPX-CAL-K25)
  • Phosphate buffered saline (PBS)
  • LifeSep 96F magnetic plate holder (Dexter Magnetic Technologies, Cat. #2501008-1)
  • 96-well clear microplates (Greiner)
  • ImageXpress® Velos™ Scanning Cytometer (Molecular Devices

Methods

Step 1. Diluted beads 1:50 in PBS and pipetted 100 μL beads into each well of a 96-well microplate.

Step 2. Read the microplate on the ImageXpress Velos Cytometer prior to washing to obtain the starting number of beads.

Step 3. AquaMax Washer microplate setup: Created a new plate in the onboard software. Started with 96-well Greiner plate, entered height as 24.2 mm (regular plate height + 10 mm LifeSep adapter height). Used the Well Depth Auto feature to determine the precise well depth and saved the resulting value in the plate definition.

Step 4. Washed plate with PBS on the AquaMax 4000 Washer. The following parameters gave the best results (minimal loss of beads during wash):

Aspirate

  • Position: Edge
  • Rate: Medium
  • Descent speed: 3
  • Height: 5 mm

Dispense

  • Volume: 150 mL
  • Rate: 5
  • Repeat 2X from step 1.
  • Aspirate (settings as above)

With the ImageXpress Velos Scanning Cytometer, fluorescence was scanned using the 532 nm laser. The number of objects detected per well before and after washing was determined using the ImageXpress Velos Cytometer software.

Pre-wash bead counts for 96-well microplate

Average number of beads per well = 615

  1 2 3 4 5 6 7 8 9 10 11 12
A 603 618 639 607 588 642 586 536 612 610 626 587
B 643 652 641 623 630 607 574 657 621 623 622 636
C 621 585 650 598 638 550 604 632 621 577 586 605
D 592 607 633 595 633 620 592 633 621 622 578 591
E 589 651 635 648 629 617 631 634 610 590 610 610
F 577 596 628 574 598 575 643 623 615 602 602 546
G 639 602 600 645 682 608 585 622 600 652 600 596
H 658 634 635 642 643 643 629 647 622 604 641 628

Post-Wash bead counts for 96-well microplate

Average number of beads per well = 585
% Retention = 95.1

  1 2 3 4 5 6 7 8 9 10 11 12
A 584 572 599 597 580 621 554 552 608 605 566 515
B 634 630 643 548 622 579 608 613 535 540 598 616
C 563 590 641 591 602 575 560 594 622 533 576 554
D 495 593 568 513 605 600 587 551 573 577 499 568
E 527 618 607 634 578 593 605 604 640 575 479 539
F 575 540 621 576 595 554 607 605 617 603 574 586
G 544 577 561 567 630 621 625 595 594 626 571 506
H 608 606 546 616 625 599 628 625 611 566 599 613

magpix beads

Figure 2. Images of pre- and post-wash representative wells. A. Pre-wash representative well: d5, 633 objects (beads) counted. B. Post-wash representative well: D5, 605 objects (beads) counted.

Conclusion

A high rate of bead retention is essential for successful MAGPIX multiplexing assays. When MAGPIX beads were washed in a 96-well microplate on the AquaMax 4000 Microplate Washer, using the LifeSep magnetic plate holder and optimized settings as described above, 95% of beads were retained in the wells. The AquaMax Washers’ highly adjustable settings and interchangeable wash heads offer the ultimate in flexibility and optimization for any assay.

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