General Immunoprofiling
Relevant Instruments:
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Leveraging antibodies against targeted proteins for visualization of specific cell types has been a long-standing practice in science for decades. While only having 3 multiplex channels, the Moxi GO II provides the easiest benchtop system for basic immunophenotyping. Moxi’s 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. The sensitivity and power of a flow cytometer can be achieved in every lab at an affordable price, which eliminates the need for delays and expensive experiments sent to a core lab.
Fluorophores
The Moxi GO II uses a 488nm laser and is equipped with a 525/40nm filter for capturing green fluorescence, plus a 561nm/LP filter for capturing orange/red fluorescence. Also 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 desirable. To the left is a list of known antibody conjugates that have been performed 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. To the left is a list of all antibodies that have been successfully tested by users on the Moxi GO II. This list is not comprehensive and is continually increasing as more users try new and different antibodies. To see a list of publications from users, please go to the ORFLO website here.
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 Moxi instrument can be exported to the computer and opened using a variety of third-party programs typically used for flow cytometry analysis. For example, 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 via this method (Fig. 2). Additional formulas and strategies to get the most information from the data can 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. A video instruction of how to perform these types of analytics can be seen here.
Summary
The Moxi GO II is a powerful benchtop instrument for obtaining 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.