General Immunoprofiling
Relevant Instruments:
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Leveraging antibodies against specific proteins for visualization of specific cell types has been a long-standing practice in science for decades. While not as robust in terms of cells counted or channels available for deep phenotyping when compared to a full-scale flow cytometer, the Moxi GO II provides the perfect benchtop option for basic immunophenotyping. Its dual orange/red and green channels allow the user to do two color analysis of their sample on thousands of cells in seconds, and the post-run analysis is similar to normal flow cytometry output data. Harness the sensitivity and power of a flow cytometer right in the lab with no need for lengthy and expensive experiments sent to the core lab!
Fluorophores
The Moxi GO II uses a 488nm laser and is equipped with a 525/40nm filter for capturing green fluorescence and a 561nm/LP filter for capturing orange/red fluorescence. Included with the Moxi GO II is an additional 646nm/LP filter that can be swapped in for the 561nm/LP filter if further red fluorophores are desired. Here is a list of known antibody conjugates that work on the Moxi GO II as well as theoretical conjugates based off of their excitation/emission spectra.
Known Antibody List
While the majority of users have utilized the Moxi GO II for immune cell identification, there are many avenues for immunoprofiling that can be taken. Here is a list of all antibodies that have been successfully been used by users on the Moxi GO II. This list is not comprehensive and is ever-increasing as more users try new and different antibodies.
Dual-Color Antibody Profiling
Fig. 1: Isolation and quantification of T-Cells in a PBMC sample using FITC-CD3 and PE-CD4. CD3- positive cells (left) CD4-positive cells (middle), and dual-positive CD3+CD4 cells (right) can be viewed in the post-run analysis.
This data was taken from a user’s freshly isolated PBMC sample to look at the T-cell population within by using CD3 and CD4 as their identification markers. Using the Moxi GO II’s intuitive post-run analysis software, it is easy to gate out debris and isolate only real cells using the particle size on the x-axis. The distinctive separation of fluorescently-positive cells on the y-axis allows for isolation of green-positive and orange/red-positive cells (Fig. 1, left, middle). Changing the x-axis to PMT1 (green channel) and the y-axis to PMT2 (orange/red channel) allows for easy identification of the dual-positive cell population, giving its concentration and the percentage of dual positive cells within the original sample (Fig. 1, right).
Fig. 2: Post-run analysis of FITC-CD3 and PE-CD4 labeled PBMCs utilizing FlowJo.
If further analysis is required, the .fcs files generated by the instrument can be exported to the computer and opened using a variety of third-party programs typically used for flow cytometry analysis. This same .fcs file used on the instrument can be easily loaded into programs such as FlowJo for more advanced analytics. The total number of events counted and the total number of dual-positive cells can be seen this way (Fig. 2). Additional formulas and strategies to get the most out of the data can also be performed, such as determining how many µL of the original sample is required to have 1,000,000 dual positive cells for a downstream experiment. An example of how to perform these types of analytics can be seen here.
Summary
The Moxi GO II is an excellent instrument for deeper levels of cell analysis due to the sensitivity of its two fluorescent channels combined with the microfluidics cassettes leveraging ORFLO’s Coulter Principle-based technology. This combination provides repeatable and reliable counting and antibody detection to give similar power to a flow cytometer at the benchtop!