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Attune NxT

What is it?

The Attune NXT (Life Technologies) is a two-laser, seven color analyzer equipped with a plate loader. The Attune NXT takes advantage of the principal of acoustic focusing to orient cells through the center of the laser intercept.

What can it do?

The Attune NXT has two lasers, 488 and 637 nm and can detect light in eight photomultiplier tubes (PMTs) and one photodiode. Seven PMTs detect fluorochromes and dyes, one PMT detects side scatter, and the photodiode detects forward scatter. Tables 1 and 2 list the common fluorochromes that the Attune's lasers excite.

Table 1: Common Fluorochromes excited by the 488 nm laser

BL1 Channel BL2 Channel BL3 Channel BL4 Channel
530/30 Filter 574/26 Filter 695/40 Filter 780/60 Filter
Alexa Fluo 488 PE PE-AF700 PE-Cy7
CF488A PI PE-Cy5.5 Qdot800
CFSE PerCP Vybrant DyeCycle Ruby
Cy2 PerCP-Cy5.5 PE-Vio770
Dyelight 488 Qdot705 VioGreen
Sytox Green PE/Dazzle 594
PerCP Vio700

Table 2: Common Fluorochromes excited by the 637 nm laser

RL1 Channel RL2 Channel RL3 Channel
670/14 Filter                   720/30 Filter 780/60 Filter
Alexa Fluor 647 Alexa Fluor 680 APC-Alexa Fluor 750
APC Alexa Fluor 700 APL-Alexa Fluor 780
Cy5 APC-Alexa Fluor 700               APC-Cy7
Qdot 655 QDot 705 APC-eFluor 450
eFluor 660 Vybrant Dye Cycle Ruby                
Sytox Red

The Attune NXT samples from 12 x 75 mm tubes, the tubes can be made of any material. The Attune Autosampler will aspirate from 96- and 384-well plates. The Autosampler works with flat, round, and V-bottom plates. Round-bottom 96-well plates generate the best results as they allow complete mixing of the sample.

The instrument tracks how much volume it aspirates making it capable of volumetric cell counting. The flow rate can be adjusted between 12.5 - 1000 µl/minute.

How does it work?

The Attune NXT aspirates sample through a 200 µm capillary. This capillary is attached to a piezo-electric device that introduces an ultrasonic wave into the capillary at a frequency ~ 2 MHz. This acoustic wave positions the cells in the sample in a single focused line through the central axis of the capillary. The Attune NXT's lasers are focused directly on the center of the stream, permitting optimal excitation of dyes and fluorochromes attached to cells. Figure 1 compares acoustic focusing and traditional hydrodynamic focusing.

diagram comparing acoustic focusing with traditional hyrdrodynamic focusingFigure 1: Comparison between acoustic focusing (A) and hydrodynamic focusing (B).

Some of the benefits of acoustic focusing are:

  1. Rapid detection of rare events. The Attune NXT runs sample up to 10x faster than conventional cytometers. The Attune NXT is capable of aspirating sample at speeds up to 1000 µl/minute and acquiring 2 x 10 7 events/sample, allowing rapid discovery of rare events.
  2. The Attune NXT is precise and sensitive at all sample rates. Acoustic focusing supports precise alignment at all speeds generating data with tighter CVs, greater sensitivity and less variation.

FAQs about the Attune NXT:

What is the laser power of the Attune NXT?
The blue 488 nm laser is 50 mW and the red 637 nm laser is 100 mW. Both lasers are flat top lasers with 50 µm laser intercepts of the stream.

How does the fluidics system of the Attune NXT work?
The sample flow rate can be adjusted between 12.5 - 1000 µl/minute. Sample delivery is accomplished by a positive displacement syringe pump. Tube sample analysis volumes can be between 20 µl and 4 ml. For the Autosampler, minimum sample volume is 50 µl in a 96-well plate with a dead volume of 30 µl. A typical 96-well plate can be run in <60 minutes with 2 wash cycles between wells to prevent carryover.

What are the performance characteristics of the Attune NXT?
The Attune NXT can reliably process data at sample flow rates up to 35,000 events/second. The Attune NXT works best with particle sizes between 0.5 - 50 µm.

What do the flashing lights on the Attune NXT indicate?
A faded blue light indicates warm up, a solid blue light means the warm up is complete. The flashing blue light means the instrument is in startup or is running an instrument function (except rinse). A solid green light indicates the instrument has completed startup and is idle. A flashing green light means the instrument is acquiring samples. A multicolor fade indicates the instrument has completed shutdown and is in sleep mode. An amber light indicates an instrument error.

Flow Cytometry Core Laboratory

University of Kansas Medical Center
Flow Cytometry Core Laboratory
Mail Stop 3050
3037 Hemenway
3901 Rainbow Boulevard
Kansas City, KS 66160