The Attune® NxT Autosampler
The Attune® NxT Autosampler, with intelligent probe design, is designed to minimize clogging and carryover (<0.5%) (Table 1) and prevent damage to the instrument. Mixing by gentle aspiration helps to ensure sample homogeneity and to maintain cell viability (Table 2), and consistent results are observed regardless of whether the sample is delivered from a tube or from a plate using the Attune® NxT Autosampler (Figure 1).
|Number of washes and % carryover
|High Throughput Mode
Table 1. Minimal carryover using the Attune® NxT Autosampler. Jurkat cells at a concentration of 1 x 106 cells/mL were dispensed into a 96-well, v-bottom plate and sampled using the Attune® NxT Autosampler. Samples were analyzed on the Attune® NxT 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® NxT Autosampler was washed 1–3 times prior to sampling the next well. Percent sample carryover was calculated.
|Percent dead cells (%)|
|Number of mix cycles||LWB||NIH/3T3|
Table 2. Increasing the number of mixing cycles on the Attune® NxT Autosampler 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, and loaded in triplicate in a 96-well, v-bottom plate. Prior to acquisition, samples were mixed 0–5 times using the Attune® NxT Autosampler, and the samples were analyzed using Standard mode collection rates (100 µL/min for NIH/3T3, 200 µL/min for LWB) on the Attune® NxT 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 mixing cycles used prior to acquisition.
Figure 1. Attune® NxT Autosampler offers 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® NxT Acoustic Focusing Cytometer equipped with a 488 nm laser for fluorescence excitation of FITC (530 BP) and R-PE (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 Standard collection rate of 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® NxT Autosampler.
Setting up an experiment with Attune® NxT 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 (Figure 2).
The Attune® NxT software is free and can be downloaded online. Multiple users can access the software—no license fees or dongles are required.
Figure 2. Designed to provide consistent well-to-well results: the Attune® NxT 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, dispensed into a 96-well, v-bottom plate, and run using a Standard collection rate of 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 percentage of propidium iodide–positive cells (dead cells) using the color gradient indicated in (A). Red-colored wells indicate 100% propidium iodide–positive cells within the sample analyzed from that well, whereas indigo-colored wells indicate a sample containing 0% propidium iodide–positive cells. The text overlaid on each well in the heat map (B) is the number 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).
|Compatible plate types||
|Minimum sample required||
|Minimum dead volume||