Software

Setting up an experiment with Attune® software is simple. Using a virtual plate layout view, you can create compensation wells, define instrument settings, and identify samples. The software also allows you to define multiple experiments on a single plate and recover unused sample (for samples collected or acquired in tube mode only).

The intuitive software is easy to use and enables quick data analysis. For example, a heat-map view allows rapid screening and confirmation of samples while the instrument is acquiring data (Figures 2 and 3).

The Attune® software is free and can be downloaded online. Multiple users can access the software-no license fees or dongles are required.

	Number of washes and % carryover
Mode	1	2	3
Standard	.01	.01	.01
High throughput	.02	.02	.02

Table 1. Minimal carryover using the Attune® Autosampler. Jurkat cells at a concentration of 1 x 10⁶ cells/mL were aliquoted into a 96-well, vbottom plate and sampled using the Attune® Autosampler. Samples were analyzed on the Attune® Acoustic Focusing Cytometer using a collection rate of Standard Mode (200 μL/min) and High Throughput (500 μL/min). Each sample was mixed once, and the Attune® Autosampler was washed 1–3 times prior to sampling the next well. Percent sample carryover was calculated.

 

Increasing the Number of Mixing Cycles Does Not Adversely Affect Cell Viability

	Percent Dead  Cells (%)
Number of Mix Cycles	LWB	NIH/3T3
0	.75	34.10
1	.78	32.83
2	.74	33.52
3	.74	32.75
4	.74	33.26
5	.75	31.58

Table 2. Gentle sample mixing using the Attune® Autosampler. Increasing the number of mixing cycles does not adversely affect cell viability. Ammonium chloride–lysed whole blood (LWB) and NIH/3T3 (live/heat-treated) cells were stained with 2 μg/mL propidium iodide, loaded in triplicate in a 96-well, v-bottom plate. Prior to acquisition samples were mixed 0–5 times by the Attune® Autosampler, and then samples were analyzed using Standard mode collection rates (100 µL/min for NIH/3T3, 200 µL/min for LWB) on the Attune® Acoustic Focusing Cytometer. Propidium iodide was excited using a 488 nm laser and fluorescence emission was collected using a 640 nm longpass filter. Minimal variation was observed in both samples, regardless of cell type and the number of mix cycles used prior to acquisition.

 

Consistent Results Regardless of Sampling Method

Figure 1. Consistent results regardless of sampling method. Whole blood lysed with ammonium chloride was labeled with mouse anti-human CD45 Pacific Orange™, mouse anti-human CD4 FITC and mouse anti-human CD8 RPE antibody conjugates. Labeled samples were analyzed on a blue/violet-configured Attune® Acoustic Focusing Cytometer equipped with a 488 nm laser for fluorescence excitation of FITC (530 BP) and RPE (574/24 BP) and a 405 nm laser for Pacific Orange™ dye (603/48 LP). Identical samples, including compensation controls, were analyzed using either (A) tube mode or (B) plate mode at a collection rate of Standard 200 μL/min. Lymphocytes were gated using a CD45 vs. side scatter plot and analyzed for expression of CD4 and CD8 antigens. Minimal variation was observed between analysis in a tube alone and on a plate running on the Attune® Autosampler.

 

Consistent Results, Well-to-Well

The Attune® Autosampler heat map function identifies variation within a parameter across a 96-well plate.

Figure 2. Consistent well-to-well results: the Attune® Autosampler heat map function identifies variation within a parameter across a 96-well plate. Live and heat-killed THP-1 cells were stained with 2 μg/mL propidium iodide, aliquoted into a 96-well, V-bottom plate, and run using a collection rate of Standard 500 μL/min with 2 mix cycles per well and 2 rinse cycles between wells. Propidium iodide was excited using a 488 nm laser (640 LP). The heat map image graphically represents the percent propidium iodine positive cells (dead cells) using the color gradient indicated in (A). Redcolored wells indicate 0% propidium iodide–positive cells within the sample analyzed from that well, whereas magenta-colored wells indicate a sample containing 100% propidium iodide–positive cells. The text overlaid on each well in the heat map (B) is the measured percentage of dead cells from each individual well within the plate. Minimal variation is observed in propidium iodide fluorescence across the entire plate, with a coefficient of variation equal to 1.44% for the entire data set (96 wells).