GeniePlex Flow Cytometer Instrument Set-Up


Instrument Set-Up Parameters

Flow cytometer set-up parameters.

Flow Cytometer Scatter Parameter cAb Bead Clustering Parameter dAb Reporter Parameter Color Compensation
BD FACSCalibur* (Single laser) FSC FL3 (670nm) FL2 (575nm) Yes
BD FACSCalibur* (Dual laser) FSC FL4 (660nm) FL2 (575nm) No
Accuri C6 FSC FL4 (660nm) FL2 (575nm) No
BD FACSCanto*, BD FACSCanto* II FSC FL4 (660nm) FL2 (575nm) No
BD FACSArray* FSC Red (660nm) Yellow (575nm) No
BD FACSVerse* (2-laser, 6-color) FSC APC (660nm) PE (586nm) No
BD CyAn ADP* FS FL8 (660nm) FL2 (575nm) No
BC FC500* FS Red2 (660nm) FL2 (575nm) Yes
Sony EC800* FS FL4 (700nm) FL2 (575nm) Yes

Note 1: The areas and heights of the FL parameters should be collected. Note 2: *Trademarks are the property of their respective owners. Note 3: For other flow cytometer set-up parameters view section titled instrument set-up protocol.

User Validated Instruments and Panels

User tested flow cytometers can be seen in the table below with the corresponding analytes in human, mouse and rat species.

Flow Cytometer Sample Type Species Analytes
BD FACSCanto II Flow Cytometer (BD Biosicence) Human serum Human IFN-γ, IL-1β, IL-1RA, IL-4, IL-6, IL-10, IL-12, IL-17, MCP-1, MIP-1α, and TNFα
BD FACSVerse Flow Cytometer (BD Bioscience) Human serum Human CCL21, CCL18 (PARC), CCL19 (MIP-3B), CCL22 (MDC), CCL27 (CTACK), CXCL2, (GROB), CXCL12 (SDF-1), CXCL16, and CX3CL1 (fractalkine)
BD FACSVerse Flow Cytometer (BD Bioscience) Human serum Human CCL21 and CCL22
BD FACSCanto II Flow Cytometer (BD Biosicence) Human plasma Human IL-1RA, MCP-1, MIP-1A, IP-10, IL-1B, IL-2, IL-6, IL-12P70, TNF-A, IFN-Gamma
BD LSR II Flow Cytometer (BD Bioscience) Rinsing liquid from dental cavities Human MMP-7, MMP-8, and MMP-9
BD Accuri C6 Plus Flow Cytometer (BD Bioscience) Human serum Human IFN-γ, IL-12p70, TNF-α, IL-2, IL-8, IL-4, IL-5, IL-9, IL-33, IL-6, IL-17A, IL-17F, and IL-22
Navios EX Flow Cytometer (Beckman Coulter) Human serum Human Inflammation 18-Plex (IFNgamma, IL-1alpha, IL-1beta, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IL-27, IL-31, IL-33, IP-10, MCP-1, MIP-1alpha, MIP-1beta, and TNFalpha) and Inflammatory Chemokine 7-Plex Panel 2 (IP-10, I-TAC, MIP-1α, MIP-1β, 6Ckine, MDC, MIP-3β)
BD FACSVerse Flow Cytometer (BD Bioscience) Human plasma Human Inflammation 11-Plex (IFNgamma, IL-1alpha, IL-1beta, IL-6, IL-8, IL-10, IL-12p70, IL-27, IP-10, MCP-1, TNFalpha)
BD FACSCanto II Flow Cytometer (BD Biosicence) Human plasma Human IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-17F, IL-22, TNF-alpha, TNFbeta, IFNgamma, IL-1RA, IL-18, G-CSF, RANTES, MCP-1, IP-10, and MIP-1 alpha
BD FACSAria I Flow Cytometer (BD Bioscience) Human plasma Human IL-10, IL-15, and IFNgamma
BD FACSCanto II Flow Cytometer (BD Biosicence) Human serum Human IL-10, and IL-35
BD FACSCanto II Flow Cytometer (BD Biosicence) Human serum Human IL-1beta, IP-10, I-TAC, MIG, IL-15, MCP-1, sCD14, IL-6, IFN-alpha2, MIP-1beta, G-CSF, and sCD163
BD FACSCalibur Flow Cytometer (BD Biosciences) Undiluted vitreous core sample Human Lipocalin2 (LCN2/NGAL)
BD FACSAria II Flow Cytometer (BD Biosciences) Cell culture supernatant Human IL-2, IL-4, IL-6, IL-10, TNFalpha, and IFNgamma
BD LSR II Flow Cytometer (BD Bioscience) Human saliva supernatant Human Inflammation 16-Plex (IFNgamma, IL-1alpha, IL-1beta, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IL-27, IL-31, IL-33, IP-10, MCP-1, MIP-1alpha, and TNFalpha)
NovoCyte Flow Cytometer (ACEA Biosicences) Human serum Human TNF-α, and IL-6
Gallios Flow Cytometer (Beckman Coulter) Human plasma Human IL-1B, IL-6, TNF-α, IL-10, CRP
BD FACSAria Fusion Flow Cytometer (BD Bioscience) Human serum Human IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL12p40, IFN-gamma, TNF-alpha, GM-CSF, MIP-1a, MIP-1b, MCP-1, and IP-10/CXCL10
BD FACSCalibur Flow Cytometer (BD Biosciences) Human serum Human IL-4, IL-5, IL-13, IL-6, IL-17A, IL-23, and TGFβ1
Gallios Flow Cytometer (Beckman Coulter) Human plasma Human IL-1B, IL-6, TNF-A, IL-10, and CRP
BD FACSCanto II Flow Cytometer (BD Biosicence) Human plasma Human IL-6, MCP-1, sCD163, VCAM-1, and adiponectin
BD FACSCanto II Flow Cytometer (BD Biosicence) Cell Culture Supernatant Human TH1/TH2/TH17 7-Plex Panel (IFNγ, IL-2, IL-4, IL-6, IL-10, IL-17A and TNFα)
BD FACScan System (BD Bioscience) Human plasma Human sCD137
BD FACSCalibur Flow Cytometer (BD Biosciences) Human serum Human IFN-γ, TNF-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12
BD FACSCalibur Flow Cytometer (BD Biosciences) Human serum Human IFN gamma, IL-2, IL-4, IL-10, IL-17A, TNFa, TGF-β1
BD FACSVerse Flow Cytometer (BD Bioscience) Bone marrow–derived hMSCs Cell cultures Human IGF-1 and IGF-2
BD FACSCalibur Flow Cytometer (BD Biosciences) Human Hofbauer cells (HBCs), macrophages of the feto-placental unit Human Custom Genieplex 23-Plex Panel
BD FACSCalibur Flow Cytometer (BD Biosciences) Human Plasma cells in culture media Human IL-1ra, IL-6 and IL-8
BD FACSCalibur Flow Cytometer (BD Biosciences) Human serum Human IL-10, IL-17, IL-35, IFN-γ
BD FACSCalibur Flow Cytometer (BD Biosciences) Cell culture conditioned medium Human FGF-2 and VEGF
BD LSRFortessa Cell Analyzer (BD Bioscience) Human tissue-conditioned medium Human IL-1β, IL-6 and IL-23p19
NovoCyte Flow Cytometer (ACEA Biosicences) Cell culture supernatant Human IFN-γ, IL-6, and TNF-α
BD FACSCanto II Flow Cytometer (BD Biosicence) Mouse serum Human AFP
Navios Flow Cytometer (Beckman Coulter) Cell culture supernatant Human ICAM-1, IL-1β, IL-6, IL-8 and MCP-1
BD LSR II Flow Cytometer (BD Bioscience) Mouse plasma Mouse IL-6, IL-10, MCP-1, and TNF-alpha
BD FACSCalibur Flow Cytometer (BD Biosciences) Mouse serum and tissue (kidney and liver) lysate Mouse TNF-α, IL-1B, IL-6, and MCP-1
BD FACSCalibur Flow Cytometer (BD Biosciences) Mouse tissue lysate samples Mouse TNF-α, IFN-gamma, IL-6, and VEGF
BD FACSCanto Flow Cytometer (BD Bioscience) Mouse cell culture supernatants Mouse IFNγ, IL-1α, IL-1β, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-23p19, IP-10, KC, MCP-1, MIP-1α, MIP-1β, RANTES and TNFa
BD Accuri C6 Plus Flow Cytometer (BD Bioscience) Mouse vaginal lavages Mouse G-CSF, IL-1α, IL-1β, IL-6, IL-10, IL17A, IL-22, IL-23p19 and TNF-α
BD FACSCanto II Flow Cytometer (BD Biosicence) Mouse bone marrow-derived dendritic cell culture supernatant Mouse IL-12, IFN-γ, IL-6, IL-1β, IL-4, IL-13
BD FACSCalibur Flow Cytometer (BD Biosciences) Mouse peripheral blood Mouse IFNγ, IL-2, IL-4, IL-6, IL-10 and IL-17A
BD FACSCalibur Flow Cytometer (BD Biosciences) Mouse serum Mouse CXCL1, CXCL2, IFN-γ, IL-6, IL-17A and TNF-α
BD FACSCalibur Flow Cytometer (BD Biosciences) Mouse serum Mouse IL-1β, IL-18, TNF-α, IL-6
BD FACSArray Bioanalyzer (BD Biosciences) Mouse mammary gland lysate and serum Mouse KC and MIP-2
BD FACSCalibur Flow Cytometer (BD Biosciences) Rat serum Rat IL-17, IL-10, and TGF-β
BD LSRFortessa Cell Analyzer (BD Bioscience) Rat serum Rat IL-4, IL-6, IL-10, and TNF-alpha
NovoCyte Flow Cytometer (ACEA Biosicences) Rat peripheral blood, penumbra, and brain tissue lysate samples Rat IL-1beta, IL-6, TNF-alpha, IFN-gamma, IL-4 and IL-10
BD FACSCalibur Flow Cytometer (BD Biosciences) Rat serum Rat IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α
BD FACSCanto II Flow Cytometer (BD Biosicence) Rat plasma Rat IL-2, IL-6, IL-10, IL-17A, and TGF-B1
BD LSRFortessa Cell Analyzer (BD Bioscience) Rat plasma Rat CCL5, CCL11, IL-4, and IL-6
NovoCyte Flow Cytometer (ACEA Biosicences) Rat plasma Rat IFN gamma, IL-1β, IL-4, IL-6, IL-10
BD FACSCalibur Flow Cytometer (BD Biosciences) Rat serum and tumor tissues Rat TNF-α and IL-1β
NovoCyte Flow Cytometer (ACEA Biosicences) Rat Kupffer cells (cell culture) Rat TNF-α, IL-1β and IL-6

Instrument Set-Up Protocol

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.

10.1 Fluorescence channels

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 Preparing instrument setup beads

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 Setting up a display layout/template

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 Running the setup beads

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.