NEWS The LC Fittings Kit is configured to connect typical eluent tubing (1.6mm or 1/16 inch OD) to the U-Series sample arm (3.2mm or 1/8 inch OD). A diagram of the kit connected to a U-Series nebulizer is shown in Figure 3. Figure 3. LC Fittings Kit Sample Tube U-Series Nebulizer LC Fittings Kit Results A prototype MicroMist nebulizer (P/N AR120-1-UFT02) combining all the benefits of the Glass Expansion U-Series nebulizer construction with the LC fittings Kit (P/N FT-16.8) was evaluated on a CyTOF2 instrument by Dr. Rahman. When designing the prototype MicroMist nebulizer, it was important to take into account the standard nebulizer operating conditions for single cell analysis by TOF-ICP-MS. In contrast to a typical ICP-MS concentric nebulizer, Mass Cytometry requires a very low nebulizer gas flow (≤ 0.3 L/min) and back pressure (≤ 20 psi) to keep the cells intact during nebulization. This poses a challenge, as with typical ICP nebulizers a low nebulizer gas flow and back pressure will produce a poor aerosol quality resulting in poor transport efficiency (poor sensitivity). Therefore, the prototype MicroMist nebulizer for single-cell analysis required a unique construction to achieve an optimal aerosol quality at a low gas flow to ensure an acceptable cell transmission efficiency. Cell transmission efficiency is defined as the proportion of single-cell (or single particle) events that are acquired by the instrument relative to the overall number of cells in the sample. When evaluating relative acquisition efficiency, it is therefore important to use matched samples and to keep the acquisition time, the cell (or particle) concentration and sample flow rate constant. For these experiments an HF resistant TruFlo monitor at the lowest range (P/N 70-803-0892) was utilized to continuously monitor the sample uptake rate. Single-cell analysis uses a very low uptake of 30-45 μL/min for most methods and maintaining a constant sample flow rate is directly related to the performance of the analysis. For this reason a syringe drive or pneumatic sample loader is used to maintain a precise sample flow rate. However, like any ICP application the nebulizer can clog, leaks can occur and the syringe barrel or pneumatic sample holder can degrade, all of which can affect the sample flow rate and analytical performance. Glass Expansion’s TruFlo sample monitor (Figure 4) provides a real-time digital display of the sample flow rate, so you always know the actual flow rate to the nebulizer. This enhances the dayto day reproducibility of results and reduces the need to repeat measurements. Figure 4. TruFlo sample monitor The gas flow rates for the prototype MicroMist nebulizer were optimized using Tuning Solution (Fluidigm), a high purity solution containing known quantities of defined elements. At a liquid flow rate of 45 μL/min, the nebulizer gas flow and make-up gas flow of the prototype MicroMist nebulizer optimized at 0.17 L/min and 0.76 L/min, respectively determined using both manual and routine autotuning protocols. These gas flow conditions match those utilized with the standard CyTOF nebulizer and provided an identical signal intensity. Nebulizer performance was then validated using EQ Calibration Beads (Fluidigm), which are polystyrene beads containing known concentrations of metal isotopes. EQ beads acquired using the prototype MicroMist nebulizer showed identical median signal intensity and CV to those acquired with the standard CyTOF nebulizer. To test performance in a typical experimental application, the prototype MicroMist nebulizer was used to acquire a stained preparation of peripheral blood mononuclear cells (PBMCs) from a healthy donor. PBMCs reflect major populations of circulating immune cells, and high dimensional analyses of these samples are a powerful tool that can be used to evaluate the immune status of patients and evaluate disease progression and treatment responses. Figure 5 shows human PBMCs that were stained with a basic immunophenotyping panel and acquired using the standard CyTOF nebulizer and prototype MicroMist nebulizer. The cell concentration (1 million cells/mL), flow rate (45 μL/min) and total acquisition time (280 sec) were kept constant. The resulting data, visualized using viSNE(15) in Cytobank, show that overall data quality, single cell staining and resolution obtained with the prototype MicroMist nebulizer were identical to the standard CyTOF nebulizer (Figure 5). The prototype MicroMist nebulizer acquired 69,827 cell events in a 280s acquisition window, providing a cell transmission efficiency of approximately 33% on the CyTOF2. www.geicp.com Glass Expansion Newsletter | Issue 41 3
GE Newsletter October 2016
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