Appnote Cell Analysis

A 30-color Immunophenotyping Panel of Mice Infected with Influenza Using the Agilent Novocyte Penteon Flow Cytometer


Use of flow cytometry has become routine across scientific disciplines, encompassing both the basic and clinical research spaces. It has proven to be a powerful tool to analyze different subpopulations of immune cells and gather a comprehensive overview of the immune system. Due to great advancements within the field, newer flow cytometers can support multiple colors, enabling identification of a variety of cells simultaneously. In this application note, a 30-color immunophenotyping panel was designed for the five-laser Agilent NovoCyte Penteon flow cytometer to see the distribution of immune cells in the lung, spleen, and mediastinal lymph nodes of mice infected with influenza.


Dimitri Popoff and Cyrille Mionnet Centre d’Immunologie Marseille Luminy, France

Camille Santa Maria and

Alejandra Felix

Agilent Technologies, Inc.


Complex and sophisticated multicolor flow cytometry experiments capable of evaluating multiple immune cells simultaneously are increasingly accessible thanks to key improvements in the field. Keeping pace with advancements, the Novocyte Penteon is equipped with five lasers (UV, 349 nm; violet, 405 nm; blue, 488 nm; yellow-green, 561 nm; and red, 637 nm) and 30 fluorescence detectors to gain the most information possible from one biological sample. With 30 colors, it is possible to identify multiple subpopulations in one tube with a single acquisition.

In this panel designed at the Centre d’Immunologie Marseille

Luminy (CIML), all detectors are used to delve into the

immune system of an influenza infected mouse and obtain

a comprehensive overview of immune cell subpopulations

post-infection. The 30-color immunophenotyping panel was

designed through diligent fluorophore selection considering

expression markers, fluorescence intensity, spectral overlap,

and Agilent NovoCyte Penteon configuration. Resulting data

was analyzed using NovoExpress software and revealed a

difference in cell distribution across three lymphoid organs.



–RPMI-1640 Medium (GIBCO, 21875034) –Fetal Calf Serum, FCS (Biowest, S1810-500) –DNAse I (Sigma-aldrich, 10104159001) –Collagenase II (Sigma-aldrich, C5138) –UltraPure 0.5 M EDTA, pH 8 (Invitrogen, 15575-038) –1X RBC Lysis Buffer (eBioscience, 00-4333-57) –FACS Buffer (PBS 1x, 0.5% BSA, 2 mM EDTA) –Brilliant stain buffer (Becton Dickinson, 563794) –Flow cytometry antibodies (Table 1) –Agilent NovoExpress Software –Agilent Novocyte Penteon flow cytometer All work was conducted on a C57Bl6 mouse, infected at

8 weeks by 5 PFU of influenza (PR8, H1N1). The mouse

was euthanized 35 days post infection. Spleen, lungs, and

mediastinal lymph nodes were extracted. A pulmonary lavage

was conducted with 10 mL of PBS 1x.

Lung and spleen: 1.Add the organ to 3 mL of RPMI 5% FCS containing 45 µL

of DNase I (150 µg/mL) and 30 µL of collagenase II (500 µg/mL).

2.Dissociate the organ tissue with OctoGentleMacs

(Miltenyi) using the "m_lung-01_02" program.

3.Wait 30 minutes at 37 °C. Run the "m_lung-01_02" program again on OctoGentleMacs (Miltenyi).

4.Add 30 µL of EDTA (0.5 M) and wait 2 minutes. 5.Filter with a cell strainer (70 µm). 6.Add media until the total volume is 15 mL. 7.Centrifuge for 7 minutes at 4 °C, 400 g. 8.Aspirate the supernatant. 9.Resuspend the pellet with 3 mL of RBC and wait

5 minutes.

10. Add media until total volume is 15 mL. 11. Centrifuge for 7 minutes at 4 °C, 400 g. 12. Resuspend in 500 µL of FACS Buffer containing 10% Fc

Block + 20% Brilliant Stain Buffer. Keep on ice.

Lymph nodes 1.Dissociate manually. 2.Filter with a cell strainer (70 µm). 3.Add media until the total volume is 15 mL. 4.Pellet the cells by centrifugation for 7 minutes

(400 g at 4 °C).

5.Resuspend the pellet in 200 µL of FACS Buffer containing 10% Fc Block + 20% Brilliant Stain Buffer. Keep on ice.

After 10 minutes on ice, count 3 million cells. Pellet cells by centrifugation for 7 minutes (400 g at 4 °C). Resuspend each sample with 50 µL of antibody cocktail (Table 1). Incubate for 30 minutes at 37 °C in the dark. Add 150 µL of FACS Buffer. Pellet the cells by centrifugation for 7 minutes (400 g at 4 °C). Rinse the cells with 200 μL of PBS.

Organ preparation

Sample preparation

Pellet the cells by centrifugation for 7 minutes (400 g at 4 °C).

Resuspend the cells in 100 µL of Live Dead Fixable

blue (diluate at 1/1,000 in PBS) for 15 minutes at room

temperature (RT) in the dark.

10. Add 150 µL of FACS Buffer. 11. Pellet the cells by centrifugation for 7 minutes

(400 g at 4 °C).

12. Acquire the samples on a NovoCyte Penteon

flow cytometer.

Table 1. Antibodies used in 30-color immunophenotyping panel on the Agilent NovoCyte Penteon Flow Cytometer, ordered by channels. The laser and filter used for detection for each antibody can be found in the table, as well as concentration and product details for each antibody.

Results and discussion

Flow cytometry has long been established as the gold standard to analyze heterogenous cell mixtures in both basic research and clinical applications. Newer, more sophisticated instruments with increased channels and lasers build upon their predecessors and expand the breadth of capabilities in lab and the clinic. By increasing the number of lasers and detectors, more antibodies can be included in a panel to identify target populations more precisely by their phenotypic markers. Immunophenotyping labels cells with fluorescent antibodies directed against cell surface markers, enabling visualization by flow cytometry. Design of a 30-color immunophenotyping panel maximizes throughput on a 30-detector instrument—to date the maximum number of detectors on a single flow cytometer.

The main objective of the panel design described in this

study was to evaluate the distribution of immune cells in

an influenza-infected mouse. Specifically, the distribution

of immune cell types across three lymphoid organs was

analyzed: lung, spleen, and lymph nodes. The subpopulations

of T cell types were also closely monitored to compare

differences in distribution across the three organs during

influenza infection. The same gating strategy was used

across all three organs to easily compare the data. Antibody

clone, detection channel, vendor information, and the amount

of each antibody added are described in Table 1.

The 30-color flow cytometry panel used in this application note identified several immune cell types across three organs by expression of cell surface receptors, as well as T cell activation status. To generate the panel, 50 µL of the antibody cocktail was added to 3 million cells. After staining, the sample data was acquired and analyzed using the NovoCyte Penteon flow cytometer and NovoExpress software. The gating hierarchy used to identify immune cell populations and subsets is described in the following paragraphs.

Initial gating with forward scatter (FSC) and side scatter (SSC)

removed debris to identify the cell population of interest.

Doublets were removed following FSC-A and FSC-H gating,

and live cells were then identified by Live/Dead Fixed Blue

Dead Cell Stain kit and carried forward for further analysis.

Hematopoietic cells were identified by expression of CD45+

and analyzed further, while low expression indicated the

presence of basophils. CD45+ cells were gated using CD19

and MHC II, which revealed a subset of B cells at high

expressions of both. B cells were gated further with IgD and

B220, which indicated approximately 61.39% of the B cells

were naïve.

Cells lacking expression of CD19 were segmented with markers for CD11b and Ly6G. High expression of CD11b and Ly6G determined that 6.23% of cells identified were neutrophils, while the rest were not. By maintaining gating for CD11b, but changing the Y axis to Siglec F, alveolar macrophages (high Siglec F, low CD11b) and eosinophils (high Siglec F, high CD11b) were identified. The population of cells with little to no expression of Siglec F were pushed forward for further dissection, as T cells contain very low levels of Siglec F.

To differentiate between T cells and others, gating for CD3+

and CD90+ was applied. CD3+CD90+ cells were then used

to closely analyze T cell subsets. CD3-CD90- cells were

further characterized to determine the remaining immune cell


NK cells were identified by gating for expression of NK1.1 and NKp46. Cells with low expression were not considered NK cells and gated against Siglec H and B220 to identify plasmacytoid dendritic cells. The rest of the cells were plotted against CD43 and Ly6C to identify monocytes. Cells not categorized as monocytes were then gated for CD88+ and CD64+ to find macrophages, which were further confirmed by gating against MHC II and CD11c. CD8-CD64- cells were similarly gated to identify dendritic cells, which populated the upper right corner of the plot. It was possible to distinguish between the two types of conventional dendritic cells, cDC1 and cDC2 by gating for XCR1, a chemokine receptor exclusively on cDC1.

T cells (CD3+CD90+ cells) were explored in further depth

than the other immune cell populations, as the objective was

to gather insight to their activation status and distribution

across the three lymphoid organs during infection.

Approximately 3.3% of the identified T cells expressed the

gamma delta receptor. The rest were gated for expression of

CD4 and CD8, then divided into two groups: CD4+CD8– and

CD8+CD4–. Gating CD4+CD8– cells for CD25 determined

less than 2% were regulatory T cells (1.66%). The rest were

gated for CD62L+ and CD44+ to distinguish between naïve

CD4+ memory cells (CD62L+) and activated CD4+ cells

(CD62L–). Resident memory T cells (Trm) were then identified

by expression of CD103+CD69. CD8+CD4- cells followed a

similar gating strategy to distinguish between CD8+ memory

(CD44+CD62L+), activated (CD44+CD62L–), and Trm

(CD103+CD69+) cells.

While Figure 1 shows data pertaining to the lung sample specifically, it is relevant across all organs, given the same strategy was used. This was done so it was easy to compare immune cell distribution and activation across all three organs. Comparison of immune cell subpopulation frequencies in the three organs revealed marked differences in distribution in some instances (Figure 2). In this study, the frequency value was the percentage parent, which refers to the percentage of the subpopulation within the population from which it comes.

A direct comparison T cell distribution across the three

organs showed there was increased T cell expression in

the lymph nodes (90%) compared to the lungs (62%) and

the spleen (54%). CD8 T cells followed a similar distribution

pattern, though much less drastic (54% vs 43%). Interestingly,

the pattern changes remarkably. Neutrophils were also

greatly reduced in the lymph nodes relative to the lungs and

spleen (1:10:20 ratio, respectively.) Meanwhile, pDC cells

had increased distribution in the lymph nodes with minimal presence in the lungs and none in the spleen. Basophils, as expected, were minimally represented in all three organs, though there was a slight uptick in distribution in the lungs relative to the other two (0.32% versus 0.01%). With 30 parameters, it is easy to have a quick overview of the organs, with a single acquisition.

The absolute count (on event/µL) of each sample was also

determined by NovoExpress Software during acquisition.

Figure 3 shows the absolute count of T cell subpopulations in

the lung of the infected mouse. A close look at the distribution

of T cells in the lung revealed the highest concentration—

measured as events per microliter—consisted of activated

CD4+ and CD8+ cells. Though not quite 1:1, the distribution

between the two did not differ greatly. The next set of most

common T cells were much less frequent. Interestingly, CD8+

Trm cells were an order of magnitude more frequent than

CD4+ Trm cells.

Figure 3. T cell subpopulation distribution according to their concentration (events/µL) in lung.


The Agilent NovoCyte Penteon flow cytometer enables researchers to study multiple important parameters in one acquisition—a great asset in immunophenotyping. With five lasers and 30 detectors, it is possible to extract more information from a single tube, saving precious biological material. Absolute count provides even more information about the sample. In this application note, the same gating strategy was applied on three lymphoid organs, permitting comparison of T cell distribution and allowing insight into the immune system during influenza infection. The high detection capabilities of the NovoCyte Penteon also allowed identification of other immune cell subsets in the lung, spleen, and lymph nodes in the same experiment

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