Electrical Detection of Innate Immune Cells

Abstract

Accurately classifying the innate immune players is essential to comprehensively and quantitatively evaluate the interactions between the innate and the adaptive immune systems. In addition, accurate classification enables the development of models to predict behavior and to improve prospects for therapeutic manipulation of inflammatory diseases and cancer. Rapid development in technologies that provide an accurate definition of the type of cell in action, allows the field of innate immunity to the lead in therapy developments. This article presents a novel immunophenotyping technique using electrical characterization to differentiate between the two most important cell types of the innate immune system: dendritic cells (DCs) and macrophages (MACs). The electrical characterization is based on capacitance measurements, which is a reliable marker for cell surface area and hence cell size. We differentiated THP-1 cells into DCs and MACs in vitro and conducted electrical measurements on the three cell types. The results showed average capacitance readings of 0.83 µF, 0.93 µF, and 1.01 µF for THP-1, DCs, and MACs, respectively. This corresponds to increasing cell size since capacitance is directly proportional to area. The results were verified with image processing. Image processing was used for verification because unlike conventional techniques, especially flow cytometry, it avoids cross referencing and by-passes the limitation of a lack of specificity of markers used to detect the different cell types.

Keywords: dendritic cells; electrical characterization; image processing; immune system; macrophages.

Figure A1

Electrochemical system optimization results for varying S_rate. (A) E_step = 0.002 and S_rate = 0.004, (B) E_step = 0.002 and S_rate = 0.04; (C) E_step = 0.002 and S_rate = 1.

Figure A2

Electrochemical system optimization results for simultaneous varying of S_rate and E_rate. (A) E_step = 0.009 and S_rate = 0.009; (B) E_step = 0.1 and S_rate = 0.1; (C) E_step = 0.01 and S_rate = 2.

Figure 1

Overlapping of cell surface markers between MACs and DCs. DCs and MACs share the same surface markers CD11c, CD11b, MHCII, CD68.

Figure 2

The two approaches for innate immune cell differentiation.

Figure 3

Average mean fluorescent intensity of different cell markers for THP-1, DCs, and MACs with S.E.M bars obtained for three measurements. Cultured cells were washed, suspended at 3 × 10⁴ in 200 µL cold FACS solution (DPBS; Gibco-Invitrogen) and incubated with FITC- or PE-conjugated monoclonal antibodies or appropriate isotypic controls for 30 min. Cells were then washed twice and resuspended in 300 µL of cold FACS solution. Stained cells were analyzed with BD Accuri C6 plus. Cell debris was excluded from the analysis by setting a gate on forward and side scatter that included only cells that are viable.

Figure 4

(A) THP-1 Immune cells before differentiation. THP-1 was first cultured in RPMI-1640 media, then differentiated into DCs and MACs. Human monocytic THP-1 cell line (ATCC, Manassas, VA, USA)35 were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate, 0.01% of mercaptoethanol, and 1% penicillin/streptomycin at 37 °C, 5% CO₂, and 95% humidity. (B) DCs and (C) MACs after differentiation, respectively. DCs were differentiated based on the Berges et al. protocol. To induce differentiation rhIL-4 (200 ng = 3000 IU/mL) and rhGM-CSF (100 ng/mL = 1500 IU/mL), rhTNF-α (20 ng/mL = 2000 IU/mL), and 200 ng/mL ionomycin were added to the FBS-free media. For the macrophages, the differentiating and activation protocols of THP-1-derived macrophages were adapted and modified from Genin et al. [37]. THP-1 cells were terminally differentiated into uncommitted macrophages (MPMA) with 300 nM phor-bol 12-myristate 13-acetate (PMA; Sigma-Aldrich, Germany) in RPMI 1640 media without FBS supplement. Afterward, cells were activated for 48 h into pro-inflammatory macrophages (MLPS/IFNγ) by adding 10 pg/mL lipopolysaccharide (LPS; Sigma, USA) and 20 ng/mL IFNγ (Biolegend, San Diego, CA, USA), or into anti-inflammatory macrophages (MIL-4/IL-13) with 20 ng/mL interleukin 4 (IL-4; Bio-legend, USA) and 20 ng/mL interleukin 13 (IL-13; Biolegend, USA). THP-1 cells have a round shape and are suspended in the media, DCs are attached and spread their dendrites in the flask. MACs are also adherent, but without the elongations of the DCs. (D–F) show the selection undertaken in ImageJ software for the calculation of the area of the THP-1, DCs, and MACs, respectively.

Figure 5

The calculated average area of each cell with S.E.M bars. MACs have the largest area, followed by DCs, and finally THP1 cells.

Figure 6

I–V curve for the three types of cells using drop sense technology. (A) THP1, (B) DCs, (C) MACs. There were no clear differences between the three graphs. RPMI full media supplemented with 10% FBS was used to dilute the cells. It was also used as the media. Measurements were conducted using a two nickel electrode configuration, scan range of −0.9 V to 0.9 V and a scan rate of 0.04 V/s.

Figure 7

Current versus time and voltage versus time curves for the three types of cells from the drop sense technology. (A) THP-1, (B) DCs, (C) MACs. RPMI full media supplemented with 10% FBS used to dilute the cells. It was also used as the media. Measurements were conducted using a two nickel electrode configuration, scan range of −0.9 V to 0.9 V and a scan rate of 0.04 V/s.

Figure 8

Capacitance–time curve for the three types of cells before media de-embedding (removing the value of media from the rest of the samples) (A) THP-1, (B) DCs, (C) MACs. Capacitance values were extracted using MATLAB, based on the fact that the capacitive current measured is proportional to the rate of change of the applied potential with the constant of proportionality equal to the capacitance. There is no consistent trend between the concentration and capacitance.

Figure 9

Capacitance–time curve for the three types of cells after media de-embedding (A) THP-1, (B) DCs, (C) MACs. De-embedding was performed by diving each of the concentration values in Figure 8 by their corresponding media value.

Figure 10

Capacitance vs. concentration for the three types of cells after the de-embedding process. (A) Capacitance versus concentration at 29.2 s where maximum capacitance occurs for each of the cell types. (B) Average capacitance for the three concentrations with S.E.M error bars. MACs had the highest values and DCs had the lowest values, consistent with the literature.