Novel Applications of Lead Acetate and Flow Cytometry Methods for Detection of Sulfur-Containing Molecules

Hydrogen sulfide (H2S) is the most recently established gaseous vasodilator, enzymatically produced from cysteine metabolism, involved in a number of pathophysiological processes. However, its accurate detection in vivo is critical due to its volatility and tendency to form sulfane sulfur derivatives, thus limiting the data interpretation of its biological roles. We developed new applications of the simple and rapid method to measure H2S release in cell culture systems, based on the lead acetate strip test. This test, previously prevalently used in microbiology, was compared with the agar trap method, applied, in parallel, on both cell cultures and cell-free samples. Sulfane sulfur represents the major species derived from intracellular H2S. Various fluorescent probes are available for quantitation of H2S derivatives intracellularly. We present here an alternative to the classic imaging method for sulfane sulfur evaluation, running on a flow cytometer, based on SSP4 probe labeling. Flow cytometry turned out to be more direct, fully quantitative and less time-consuming compared to microscopy and more precise with respect to the fluorescence multi-plate reader assay. The new application methods for H2S determination appear to be fully suitable for the analysis of H2S release and sulfane sulfur content in biological samples.


Introduction
Hydrogen sulfide (H 2 S), previously only considered a hazardous gas, was, in the 1990s, detected in mammalian tissues and found indispensable in physiological concentrations. H 2 S is biosynthesized through enzymatic reactions in human tissues, although 50% of this gas is generated by the microbiota, in the inner and outer mucus layers of the intestine [1]. In the last two decades, the metabolism and physiological roles of H 2 S and its derivatives, such as the amount released from Fe-S proteins and sulfane sulfur species, have been also related to various pathological situations [2,3]. Controversies have been raised on the physiological H 2 S active concentrations, which have been detected in the 10 −5 −10 −4 molar range [4,5]. Detailed study of H 2 S role in metabolic processes showed its beneficial effects on neuromodulation [6], cardiovascular (CV) [7][8][9] and immune systems, while its impairment

Measuring of Sulfane Sulfur Species Using SSP4 and Flow Cytometry in Comparison to Fluorescence Microscopy
The aim of this part of the experimental design was to optimize the use of SSP4 as a fluorescence probe to quantitate sulfane sulfur intracellularly. SSP4 is a fluorescent probe of the last generation, capable of reacting with sulfane sulfur species, according to the reaction scheme in Figure 2a, thus allowing quantitative detection with high accuracy, reproducibility and sensitivity. Fluorescence microscopy and spectrometry (including multi-plate readers) are the recommended techniques to detect SSP4 labeling, as suggested by the supplier's protocol. Our aim was to optimize this technique, by increasing precision and dependability of this approach. We chose flow cytometry to evaluate sulfane sulfur content in live cells using SSP4, in parallel fluorescence microscopy for comparison ( Figure 2b). In addition to the experimental groups described in Section 2.1, we accomplished these experiments under various conditions. We included in the protocol cell samples treatments with glutathione (GSH) and stimulators (Cys, B6, and SAM), which are expected to increase sulfane sulfur formation. In fact, it was demonstrated that activation of CBS/CSE, in presence of GSH and Cys, generates CysSSH and CysSSSH, subsequently forming sulfane sulfur species due to sulfur transfer from CysSSH to GSH in a CBS/CSE dependent manner [42]. Fluorescence microscopy and spectrometry (including multi-plate readers) are the recommended techniques to detect SSP4 labeling, as suggested by the supplier's protocol. Our aim was to optimize this technique, by increasing precision and dependability of this approach. We chose flow cytometry to evaluate sulfane sulfur content in live cells using SSP4, in parallel fluorescence microscopy for comparison ( Figure 2b). In addition to the experimental groups described in Section 2.1, we accomplished these experiments under various conditions. We included in the protocol cell samples treatments with glutathione (GSH) and stimulators (Cys, B6, and SAM), which are expected to increase sulfane sulfur formation. In fact, it was demonstrated that activation of CBS/CSE, in presence of GSH and Cys, generates CysSSH and CysSSSH, subsequently forming sulfane sulfur species due to sulfur transfer from CysSSH to GSH in a CBS/CSE dependent manner [42].     Prepare flasks for agar trap method as described before [3,17]. Flasks are laid horizontally upside-down; 5 mL of 1% agar with 7.23 mM ZnAc and 162 mM NaOH are allowed to polymerize on the bottom surface inside each flask. Once the agar is solidified, turn the flasks and plate cells on the bottom. Treat cells as appropriate. At the end of treatment, withdraw the medium and lay 2 mL of 500 mM NaOH, 100 µM EDTA solution onto the agar gel and right after 500 µL of detection solution (17.1 mM NDPS and 37 mM FeCl 3 in 6 M HCl) [43]. Measure color development in 15 min with spectrophotometer at λ668 nm against an agar gel blank kept with cell free DMEM (Figure 3a Cut lead acetate strips using a paper perforator; 2.
Place test strips into the internal side of each flask lid, using a double-sided adhesive tape.

3.
Sterilize flask lids using UV light. Perform LAST and agar trap experiments, in triplicates in two biological repeats. Each sample consist of 500,000 cells in 5 mL of DMEM left overnight before treatment in 25 mL cell culture flasks. Standard treatment duration is 6 h and 12 h, respectively, under the above conditions.

Agar Trap Method
Prepare flasks for agar trap method as described before [3,17]. Flasks are laid horizontally upside-down; 5 mL of 1% agar with 7.23 mM ZnAc and 162 mM NaOH are allowed to polymerize on the bottom surface inside each flask. Once the agar is solidified, turn the flasks and plate cells on the bottom. Treat cells as appropriate. At the end of treatment, withdraw the medium and lay 2 mL of 500 mM NaOH, 100 μM EDTA solution onto the agar gel and right after 500 μL of detection solution (17.1 mM NDPS and 37 mM FeCl3 in 6 M HCl) [43]. Measure color development in 15 min with spectrophotometer at λ668 nm against an agar gel blank kept with cell free DMEM (Figures 3a,d, 4a and 5). For standard curve, add 3 mL aqueous solution containing 25, 50, 100, and 200 µM NaHS to flasks and tightly close with the strip containing lids, following incubation for 1 h at 37 • C.

5.
At the end of the experiment, place test strips on Whatman paper sheets, and scan using a multifunctional printer. Perform LAST and agar trap experiments, in triplicates in two biological repeats. Each sample consist of 500,000 cells in 5 mL of DMEM left overnight before treatment in 25 mL cell culture flasks. Standard treatment duration is 6 h and 12 h, respectively, under the above conditions.

Agar Trap Method
Prepare flasks for agar trap method as described before [3,17]. Flasks are laid horizontally upside-down; 5 mL of 1% agar with 7.23 mM ZnAc and 162 mM NaOH are allowed to polymerize on the bottom surface inside each flask. Once the agar is solidified, turn the flasks and plate cells on the bottom. Treat cells as appropriate. At the end of treatment, withdraw the medium and lay 2 mL of 500 mM NaOH, 100 μM EDTA solution onto the agar gel and right after 500 μL of detection solution (17.1 mM NDPS and 37 mM FeCl3 in 6 M HCl) [43]. Measure color development in 15 min with spectrophotometer at λ668 nm against an agar gel blank kept with cell free DMEM (Figures 3a,d, 4a   CRITICAL STEP Scanning should be performed within 24 h after experiment, since, within 3-5 days after experiment, the color can fade away. 6. Image analysis is accomplished using ImageJ software (National Institutes of Health, Bethesda, MD, USA): Analyze > Measure and Analyze > Histogram. Set fixed reading slit for each series of assay measurements (see Figure 3b,d).
LAST in Cell Culture. Time for Completion: Four Days 1.
Treat cell culture in flasks as described above and tight lids containing test strips. Incubate for 6 h and/or 12 h in 5% CO 2 atmosphere.

2.
At the end of experiment, repeat Steps 5 and 6 as in the previous paragraph.

3.
Data are analyzed with ImageJ software, using GraphPad Prism 5 for statistics (GraphPad Software, La Jolla, CA, USA) and applying Student's two-tailed t-test (Figure 4b).

Sulfane Sulfur Quantitation with SSP4 Using Flow Cytometry in Comparison to Fluorescence Microscopy. Time for Completion: 00:45 Hours
Plate 60 mm petri dishes with 500,000 cells, in 3 mL of DMEM and leave overnight in incubator, before the experiment. Prepare enough dishes to perform each treatment for both fluorescence microscopy and flow cytometry in triplicate in two biological repeats, during 3 h.

Sulfane Sulfur Quantitation with SSP4 Using Fluorescence Microscopy
Follow the general directions reported in the technical manual provided by the supplier of the SSP4 fluorescent probe [44]. Briefly, after 3 h treatment, wash each cell sample twice with PBS and incubate for 15 min in 2 mL of FBS-free DMEM, with 10 μM SSP4 and 500 μM CTAB. Subsequently, wash once again and leave in PBS for imaging. SSP4-labeled cell imaging is captured using a confocal laser scanning microscope at λex482 nm and λem515 nm. For data analysis, employ the ImageJ software ( Figure 6).   (c) microphotograph of agar gel in the sample incubated at 36% of humidity; and (d) microphotograph of agar gel with microdrops in a sample incubated at 62% of humidity. p value versus sample incubated at 36% of humidity: *** p < 0.001 (according to the Student's two-tailed t-test).

Sulfane Sulfur Quantitation with SSP4 Using Flow Cytometry in Comparison to Fluorescence Microscopy. Time for Completion: 00:45 Hours
Plate 60 mm petri dishes with 500,000 cells, in 3 mL of DMEM and leave overnight in incubator, before the experiment. Prepare enough dishes to perform each treatment for both fluorescence microscopy and flow cytometry in triplicate in two biological repeats, during 3 h.

Sulfane Sulfur Quantitation with SSP4 Using Fluorescence Microscopy
Follow the general directions reported in the technical manual provided by the supplier of the SSP4 fluorescent probe [44]. Briefly, after 3 h treatment, wash each cell sample twice with PBS and incubate for 15 min in 2 mL of FBS-free DMEM, with 10 µM SSP4 and 500 µM CTAB. Subsequently, wash once again and leave in PBS for imaging. SSP4-labeled cell imaging is captured using a confocal laser scanning microscope at λ ex 482 nm and λ em 515 nm. For data analysis, employ the ImageJ software ( Figure 6).

Agar Trap Method
Prepare flasks for agar trap method as described before [3,17]. Flasks are laid horizontally upside-down; 5 mL of 1% agar with 7.23 mM ZnAc and 162 mM NaOH are allowed to polymerize on the bottom surface inside each flask. Once the agar is solidified, turn the flasks and plate cells on the bottom. Treat cells as appropriate. At the end of treatment, withdraw the medium and lay 2 mL of 500 mM NaOH, 100 μM EDTA solution onto the agar gel and right after 500 μL of detection solution (17.1 mM NDPS and 37 mM FeCl3 in 6 M HCl) [43]. Measure color development in 15 min with spectrophotometer at λ668 nm against an agar gel blank kept with cell free DMEM (Figures 3a,d, 4a and 5 (Figure 4b).
CRITICAL STEP The subsequent analysis should be performed within 5-15 min.

5.
Analyze data using Accuri™ C6 Plus and GraphPad Prism 5 software (Figure 7).   Color code is the same in (a-c) and the inset of (d).

Expected Results
The lead acetate strip test was successfully adapted to quantify H 2 S release in cell culture and, compared with agar trap, was equally sensitive, during the first hour of incubation. Standard curves, for the agar trap and LAST methods, showed linear or exponential dependence of the amount of H 2 S released as a function of NaHS concentration (Figure 3a,b, respectively). The sensitivity of the two methods appeared comparable. In these experiments, NaHS, used as H 2 S donor, was dissolved in an aqueous solution and, hence, released as H 2 S is measured.
The estimated detection linear range for gaseous H 2 S, released in 1 h at 37 • C from an aqueous solution of NaHS, using LAST method falls within 3-3.5 µg and 56 µg of NaHS. In terms of concentration, this corresponds, in good approximation, to 10÷200 µM NaHS initially dissolved in aqueous solutions. For LAST, we used 4 mm Ø lead acetate treated filters. Thus, the minimal amount of sulfur containing solute to be detected was 0.28 µg to be absorbed onto 1 mm 2 filter surface. Saturation was reached at 4.5 µg/mm 2 . Agar trap method displays similar performances compared to LAST with respect to the lower threshold sensitivity level. Conversely, saturation was reached at lower NaHS amount (about 45 µg), corresponding to 160 µM NaHS in solution. We figured out that sensitivity of LAST and agar trap methods could be further increased by 25%, by reducing surface area of the lead acetate filter and by reducing gel surface, thus optimizing detection solution volume, respectively.
On the other hand, according to Sitdikova and coworkers [45], a strict relationship exists between the initial NaHS concentration and that of H 2 S released from the NaHS donor, by plotting the NaHS donor vs. the H 2 S currently measured with an anion selective electrode. According to this extrapolation, it was possible to indirectly evaluate H 2 S by multiplying NaHS concentration for a 0.4 factor. Applying this algorithm, the relationship of H 2 S concentrations derived from NaHS donor was linear for both agar trap and LAST methods, up to about 40 µM H 2 S, corresponding to 100 µM NaHS. The established range of linear dependence is suitable for experiments with cell cultures. In fact, in previous work, our research group observed maximal H 2 S release from stimulated hepatocarcinoma cells (HepG2) in the 30-50 µM range [3]. Standard curves resulting from NaHS were reproducible and linear, R 2 = 0.99 (Figure 3c), which allowed us to proceed with LAST analysis in cell culture.
The following analysis of H 2 S release from cell culture showed consistent results for agar trap and LAST methods within 6 h of the various cell treatments (see Figure 4). In both experiments, CTRL (+) and DATS with stimulators in cell culture significantly increased the amount of released H 2 S in comparison to intact samples (dashed line) or in presence of CBS/CSE inhibitors. However, the LAST method resulted in a more precise and less dispersed data, after statistical analysis.
Importantly, agar trap method seemed to diminish its sensitivity in experiments lasting longer than 6 h. We noticed 10-fold lower absorbance unit range in the 12 h experiment (inset in Figure 4a) in comparison to the experiment with 6 h incubation (Figure 4a). LAST and agar trap showed quite different results after longer cell exposures (12 h, insets in Figure 4). Noteworthy, the ratio of H 2 S levels, among samples detected by agar trap in 12 h, remained similar to that detected after 6 h (Figure 4a and inset in Figure 4a, respectively). DATS is a H 2 S donor that could induce saturation, while stimulators maintained CBS/CSE activity for much longer. Thus, levels of H 2 S released within 12 h in CTRL (+) and in DATS with stimulators become very similar, according to LAST (inset in Figure 4b), as opposed to results obtained with the agar trap method (inset in Figure 2a). We hypothesize that LAST, although capable of providing a result in a shorter incubation time, is also more suitable than the agar trap method to quantitate H 2 S release in long-lasting experiments (over 6 h), in that it does not appear to reach saturation within this time range. As shown in Figure 5a, the percentage of humidity significantly (≥ 28%) altered the agar trap sensitivity. It was noticed that agar layer in the flask with cell culture, in the CO 2 incubator, became covered with condensation drops (Figure 5d). This did not occur in a lower humidity microenvironment (Figure 5c). Thus, the LAST method appears to be superior with respect to the agar trap, to measure H 2 S release from cell cultures, in that it insures: (a) higher stability over time; (b) less tendency to saturation of the detection system; and (c) does not appear to be crucially influenced by a high degree of humidity in the chamber.
Both agar trap and LAST allow the detection of the total quantity of H 2 S released over time, during cell incubation in contrast to ultra-sensitive methods such as pre-column derivatization with monobromobimane and HPLC analysis (MBB/HPLC), able to detect H 2 S at nanomolar levels. For example, MBB/HPLC is a destructive method and does not allow the recovery of H 2 S extracted and entrapped in a filter throughout the duration of the experiment. We may say that the two methods are complementary, in the sense that LAST may provide a "rule of thumb" best approximation of H 2 S total release during incubation. Conversely, other more sensitive methods are suitable to detect end-point fine-tuned H 2 S release, provided that incubation time and experimental conditions are suitable to prevent massive evaporation of H 2 S, or other ways to entrap this gas are employed.
Moreover, LAST does not need any special laboratory preparation or equipment. It is rapid, and cell samples are not consumed in the assay and can be recovered and used for further determination. The optimal range of released H 2 S that can be detected by LAST assay corresponds to H 2 S physiological concentrations found in mammals [4,5].
The common methods to quantify sulfane sulfur derivatives, employing fluorescent probes, are accomplished by fluorescence microscope imaging [35,42,46] or fluorescence spectrometry [14,36,42,[47][48][49][50]. Fluorescence microscopy may achieve fully quantitative performances, by measuring fluorescence intensity (FI) using ImageJ or similar software. The spectrofluorometer (or multi-plate reader) also allows evaluating FI quantitatively, but this is performed on an approximate cell count based on the assumption of cell confluence in a certain plate surface. Sulfane sulfur detection, performed according to user's manual supplied with SSP4, requires spectrofluorometer or multi-plate reader. Following the original protocol, we noticed the high divergence in quantity of cells which were lost during procedures. The alterations of cells adhesion and consequently their loss after washing procedures can be due to CTAB, a cationic surfactant hampering cell adhesion [51]. CTAB however is needed to transport SSP4 into the cells. We improved the original protocol, by reducing both SSP4 concentrations used, along with cytotoxic CTAB concentrations, the latter from 500 to 150 µM. These changes result in a more stable cell count during FI detection, which significantly increases reliability of results.
Flow cytometry allows exact quantitation of FI on a single cell basis and is considered to be superior, by accuracy, compared to FI evaluation by confocal microscopy or multi-plate reader analysis. In fact, the data obtained using confocal microscope ( Figure 6) and using flow cytometer (Figure 7) display some crucial differences: (a) The data obtained with flow cytometer are much more precise: the quantity of reads is identical for each sample (3000 reads), resulting in a negligible SD, indeed. (b) The quantity of sulfane sulfurs in cells treated with GSH plus stimulators is over 20% higher than that detected in stimulated controls, CTRL (+), while no significant difference can be detected by microscopy.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.