Flow cytometry analysis

The viability, cell morphology, and expression of cell surface activation markers after TiO2

stimulation was determined with flow cytometry. Flow cytometry is a technique that allows to detect size, granularity, and fluorescent signals of cells that have been tagged with fluorescently labelled antibodies against specific molecules, often cell-surface antigens. In the flow cytometer the stained cells pass through a laser beam in the flow cell, and the scattered light as well as any fluorescent signal emitted by the cells are digitally recorded by

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photomultiplier tubes (PMTs). The detection of a signal by the detectors is called an event. Scattered light is recorded by the forward scatter (FSC) and side scatter (SSC) detectors. The FSC signal is dependent on the size of the cell, and the SSC signal corresponds to the granularity of the cell. Larger cells have higher FSC values compared to smaller cells, and cells with a high level of internal complexity, e.g. through the presence of granules, have a high SSC profile. In addition, each fluorescent detector records emitted light of a specific wavelength. Cells that highly express a specific antigen which has been fluorescently labelled by incubation with an antigen-specific antibody will show high fluorescence values in the respective detection channel. The advantage of flow cytometry is that it is a high throughput method because several thousand events can be detected per second. Furthermore, several characteristics can be measured at the same time.

For the flow cytometry experiments BMDMs were first stimulated with TiO2 suspension

without or with MDP or PGN for 3 h or 3 h + 21 h. Then, the stimulated BMDMs were incubated in cold phosphate-buffered saline (PBS; Life Technologies) for 30 min on ice and collected by vigorous pipetting. The cells were resuspended in 150 µL cold fluorescence-activated cell sorting (FACS) buffer, which consisted of PBS containing 2 % FBS, 1 mM ethylenediamine tetra-acetic acid (Life Technologies), and 0.01 % sodium azide (BDH Laboratory Supplies, Poole, UK). Next, the BMDMs were incubated with 1 µg/mL anti-mouse CD16/32 fragment crystallisable region receptor blocking antibody (clone 93; BioLegend, San Diego, CA, USA) for 15 min on ice to prevent unspecific binding of the fluorescently labelled antibodies. Then, the cells were incubated with fluorescently labelled antibodies (all from Biolegend) for 15 min on ice in the dark. The anti-mouse antibodies used for the staining included a marker for murine macrophages, namely phycoerythrin-labelled F4/80 (clone BM8), and Alexa Fluor 488/647-labelled antibodies for the detection of the cell surface activation molecules CD86 (clone GL-1) and CD80 (clone 16-10A1), respectively. The antibodies were used at a concentration of 1 µg/mL for F4/80, or 2.5 µg/mL for both CD80 and CD86.

Titration of the antibodies had shown that at these concentrations the fluorescent signal in relation to the amount of antibody used was adequate. Briefly, for the titration 1 × 106 splenocytes from WT mice were incubated with the respective antibodies in concentrations representing 6-fold serial dilutions from 1:50 to 1:1600 relative to the original antibody concentration. Then, the percentage of cells that stained positive with the respective antibody were compared between the dilution steps. The initial concentrations, which represented a dilution of 1:50 relative to the original antibody concentration, were 4 µg/mL for F4/80 and

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10 µg/mL for CD80 and CD86. It was observed that the fluorescent signal at a dilution of 1:200 was similar to the signal obtained at a dilution of 1:50 for all antibodies (data not shown). Therefore, all antibodies were used at a dilution of 1:200 in the subsequent flow cytometry experiments, which corresponded to the antibody concentrations mentioned in the previous paragraph.

Immediately prior to analysis, 0.8 µg/mL propidium iodide (PI; Life Technologies) was added to each sample to be able to assess cell viability through PI exclusion. The stained cell suspensions were analysed with a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) with a 488 nm argon-ion laser and a 635 nm red-diode laser. With this type of flow cytometer four different fluorescent signals can be detected at the same time in addition to FSC and SSC signals. At least 12,000 total events per sample were acquired with the CellQuest Pro software version 6.0 (BD Biosciences), and the data were analysed with FlowJo version 9.4.3 (Tree Star, Ashland, OR, USA). The gating strategy for the analysis of the flow cytometry data is shown in Figure 2.1. The first step of the analysis was to exclude cell fragments by selecting only events with a FSC intensity greater than 200. Then, the cell viability was determined by separating events based on their PI fluorescence. PI is a red fluorescent dye that binds to nucleic acids and can only penetrate cells with damaged plasma membranes. Therefore, PI staining can be used to assess cell viability because only dead cells will incorporate PI and show positive staining. PI that is bound to nucleic acids can be excited by the 488 nm laser and will emit detectable light in the red spectrum. Thus, live cells can be distinguished from dead or apoptotic cells by flow cytometry. All events with a PI fluorescence less than 101 _{on a logarithmic scale were considered PI negative (PI}−_{) and} therefore viable. Next, the PI− _{events that were also F4/80 positive (F4/80}+_{) were selected} for further analysis. Events with a F4/80 fluorescence greater than 101 _{on a logarithmic scale}

were considered F4/80+_{. Fully differentiated BMDMs were expected to express F4/80, a}

general marker for murine macrophages [265].

The parameters that were subsequently investigated from this PI− _F4/80+ _{cell population}

were the median intensities of the FSC, SSC, CD80, and CD86 signals. The measurements of FSC and SSC signals have been used previously to assess TiO2 uptake of cultured cells [264, 266]. In addition, the SSC ratio was determined by dividing the median SSC

intensity of TiO2-treated BMDMs by the median SSC intensity of the control cells of the

respective genotype [266]. The median fluorescence intensity (MFI) signals of the co-stimulatory molecules CD80 and CD86 were also evaluated. These cell-surface molecules bind to the receptors CD28 or cytotoxic T-lymphocyte antigen 4 (also known as CD152) on

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Figure 2.1 Gating strategy for flow cytometry analysis of TiO2-exposed BMDMs.

TiO2-exposed BMDMs were analysed by flow cytometry. At least 12,000 events were acquired per sample.

(a) All acquired events with a FSC intensity greater than 200 were selected for further analysis. (b) Fluorescence of PI against FSC intensity was analysed. Events were considered PI− _{when the PI fluorescence was less}

than 101_{. (c) F4/80 expression against FSC was assessed for all PI}− _{events. Events were considered F4/80}+

when the F4/80 fluorescence was greater than 101_{. Shown are representative frequency dot plots of BMDMs}

incubated for 3 h in TCM. A colour range from blue to red is used to indicate the frequency of dots on the same spot; blue represents a single event, red represents most events. The pink lines indicate the gates with the percentage of events in the respective gate shown in the top corners.

FSC SSC PI FSC F4/80 FSC

a

b

c

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T cells [267-269]. Interaction of the co-stimulatory molecules with CD28 is required for T cell activation by APCs [270]. Thus, assessing the expression of CD80 and CD86 on BMDMs after treatment with TiO2 particles may inform on the ability of these cells to stimulate immune responses by effector immune cells. All treatments for the flow cytometry analysis were performed in triplicate and the experiment repeated once.

2.3.7

Exposure of human peripheral blood mononuclear cells to