GeniePlex Flow Cytometer Instrument Set-Up

To view full protocol visit pages 21-24 of the technical manual and see below for section 10 which details the set-up parameters for flow cytometers.

The maximum emission of the bead classification dye is at 700 nm. It can be detected on “PE-Cy5“ channels of most of the flow cytometers with blue (488 nm) excitation. It can also be detected on PE-Cy7 channels with blue (488 nm) excitation or APC channel with red (633 or 640nm) excitation if such a florescence channel is available.

The reporter dye of the GeniePlex assays is PE and can be detected on the PE channel with
blue (488 nm) excitation.

10.2.1 BLANK BEADS: Aliquot half (e.g. 75 µL if resuspension volume in Step 9.29 is 150 µL) of bead suspension from one of Blank wells from Step 9.32 into a sample tube or a well of a 96-well plate depending on the sample loading mechanism of a flow cytometer. Add 100 to 300 µL of 1x Reading Buffer to the tube/well.

NOTE: Remaining capture bead working suspension from Step 9.3 can also be used for this purpose. Aliquot 45 µL of the remaining capture bead working suspension from Step 9.3 into a sample tube or a well of a 96-well plate depending on the sample loading mechanism of a flow cytometer. Add 100 to 500 µL of 1x Reading Buffer to the tube/well.

10.2.2. STANDARD 1 BEADS: Aliquot half (e.g. 75 µL if resuspension volume in Step 9.29 is 150 µL) bead suspension from one of the Standard 1 wells from Step 9.32 into a sample tube or a well of a 96-well plate depending on the sample loading mechanism of a flow cytometer. Add 100 to 300 µL of 1x Reading Buffer to the tube/well.

NOTE: Add 75 µL - 150 µL of 1x Reading Buffer to the Blank and Standard 1 wells. Acquisition for both wells will be slower (less bead concentrations) during the sample acquisition step. IMPORTANT: When running a panel the first time, we recommend running one extra well of Standard 1 to provide proper instrument setup prior to running all standards and samples.

10.3.1. Perform instrument start up and verification of fluidic stability and optical alignment by following cytometer manufacturer‘s recommendations including running calibration and/or performance beads, such as Spherotech’s Rainbow Calibration Particles (Rainbow Calibration Particles.pdf).

10.3.2. Open a new protocol.

10.3.3. Create the following plots and histograms:

1) A dot plot with FS (X-axis) and SS (Y-axis) in linear display mode.
2) 2 histograms of “ PE-Cy5“ in Log display mode.
3) 2 dot plots with PE (X-axis) and “PE-Cy5“ (Y-axis) in Log display mode.
4) If PE-Cy7 or APC channel is available, create 2 histograms of “PE-Cy5“ in Log display mode and 2 dot plots with PE (X-axis) and “PE-Cy7“ or “APC“ (Y-axis) in Log display mode.

10.3.4. Set all compensation to zero.

10.3.5. Both Areas and Heights, if available, of the FL parameters should be collected.

10.3.6. Save the protocol.

10.4.1. Run the BLANK BEADS prepared in Step 10.2.1.
1) Adjust FS and SS gains so that the bead populations are on scale (Figure 1).
2) Create Gate 1 for the smaller (4-micron size, S4) beads and Gate 2 for the larger (5-
micron size, S5) beads (Figure 1).

3) Gate 1 to one of the “PE-Cy5“ histograms and one of the PE/“PE-Cy5“ dot plots.
4) Apply Gate 2 to the other “PE-Cy5“ histogram and the other PE/“PE-Cy5“ dot plot
5) Adjust “PE-Cy5“ PMT voltage so that all the bead populations are clearly separated on the histograms and dot plots (Figure 2).
In this example, S4 has 4 bead populations, S4P3, S4P7, S4P9 and S4P11. S4P3 (Size 4-micron, Peak #3) is the dimmest and S4P11 (Size 4-micron, Peak #11) is the brightest.
6) Adjust PE PMT voltage so that the dimmest bead population is positioned within the first decade on the PE-axis of the plot (Figure 2).
7) Save the protocol.

10.4.2. Run the Standard 1 beads prepared in Step 10.2.2.
1) Verify all the bead populations on the PE-axis are on scale (Figure 3).
2) Adjust PE PMT voltage if needed. If adjustment is needed, make sure re-run the
Standard 8 and/or make sure the dimmest bead population is still visible on the PE/“PECy5“ dot plots.

3) Apply proper “PE-Cy5“ - %PE color compensation so that the bead populations are in a horizontal position (Figure 4: small blue circle is an example of proper color compensation setting).

Over-compensation should be avoided (Figure 5: large blue circle is an example of over-compensation).

4) Save the protocol.

Note: If PE-Cy7 or APC fluorescence channel is available, carry out Steps 10.4.1 and 10.4.2 for the PE-Cy7 or APC channel for the proper PMT voltage and color compensation (usually zero) settings.