Spatially resolved quantification of oxygen consumption rate in ex vivo lymph node slices

Cellular metabolism has been closely linked to activation state in cells of the immune system, and the oxygen consumption rate (OCR) in particular serves as a valuable metric for assessing metabolic activity. Several oxygen sensing assays have been reported for cells in standard culture conditions. However, none have provided a spatially resolved, optical measurement of local oxygen consumption in intact tissue samples, making it challenging to understand regional dynamics of consumption. Therefore, here we established a system to monitor the rates of oxygen consumption in ex vivo tissue slices, using murine lymphoid tissue as a case study. By integrating an optical oxygen sensor into a sealed perfusion chamber and incorporating appropriate correction for photobleaching of the sensor and of tissue autofluorescence, we were able to visualize and quantify rates of oxygen consumption in tissue. This method revealed for the first time that the rate of oxygen consumption in naïve lymphoid tissue was higher in the T cell region compared to the B cell and cortical regions. To validate the method, we measured OCR in the T cell regions of naïve lymph node slices using the optical assay and estimated the consumption rate per cell. The predictions from the optical assay were similar to reported values and were not significantly different from those of the Seahorse metabolic assay, a gold standard method for measuring OCR in cell suspensions. Finally, we used this method to quantify the rate of onset of tissue hypoxia for lymph node slices cultured in a sealed chamber and showed that continuous perfusion was sufficient to maintain oxygenation. In summary, this work establishes a method to monitor oxygen consumption with regional resolution in intact tissue explants, suitable for future use to compare tissue culture conditions and responses to stimulation.


Supplemental Figures
Correlation plot between the initial O2 concentration and the initial rate of O2 consumption in live tissue slices, for the data shown in Figure 4e.The strength of a linear association between the two variables was measured using a two-tailed Pearson correlation coefficient test, and the R squared value of 0.14 indicated no correlation between the variables.

Captions for supplemental movies
Movies showing fluorescent signal from PDTFPP oxygen sensor overlaid with live and killed lymph node tissue slices.Images were taken every 5 seconds for a total of 5 minutes.PdTFPP signal is in cyan color and B220 (B cell biomarker) is in magenta color.Three movies are provided; two representative of live slices and one for killed slices.Table S1: Fluorescent antibodies used to label cells for fluorescence microscopy.

Dissolved oxygen calculations and assumptions: Converting mmHg to mM O2
Dissolved oxygen concentration in solution is affected by factors such as atmospheric pressure and the solubility of O2(g) in the liquid, which is inversely proportional to solvent temperature and ionic strength.
To calculate the solubility of oxygen gas in PBS, we used Henry's law of solubility (Eq.S 1).This Law provides a mathematical description of gas solubility in a liquid medium.According to Henry's law, at a constant temperature, the solubility of a gas in a liquid is directly proportional to the partial pressure of the gas above the liquid at equilibrium.
Where C is the solubility of a gas at a fixed temperature in a particular solvent (mM), H is Henry's law constant (mmHg/ mM), and ∂Pgas is the partial pressure of the gas (mmHg).The Henry's law constant for a solution is dependent on the concentration of electrolytes and proteins, atmospheric pressure, and temperature of the solution.Here, we assumed a standard atmospheric pressure of 760 mmHg and temperature of 37 °C for all experiments.Ionic strength of the 1x PBS used here (LONZA; Catalog No: 17-516F) was calculated to be 166 mM at pH 7.4.
Next, we determined the partial pressure of O2 in the cell culture incubator and in the stage-top chamber used for the optical assay.At 37 °C and 100% humidity (inside incubator), water vapor exerts a partial pressure of 47 mmHg. 1 Therefore, water vapor will make up 6.2% of the total gas at sea level (47 mmHg/ 760 mmHg).To calculate the final O2% in the gas phase under different conditions used for cell culture, we used the following equation: 3][4] Henry's constant under these known conditions can be solved as follows: • Incubator without 5% CO2 H = ∂Pgas/C = 149.7 mmHg / 0.195 mM; H = 767.69mmHg/mM Therefore, we used 767.69 mmHg/mM as the Henry's constant for conversions between mmHg to mM O2 throughout the manuscript.

Shear stress calculation for flow of PBS in perfusion chamber
To estimate the fluid shear stress (FSS) resulting from the flow of 1x PBS through the perfusion chamber, we assumed a thin rectangular cross section (Eq.S 3) 5 :

FSS = (6ղQ) / (h 2 × w)
Eq. S 3 Where ղ is fluid viscosity, Q is fluid flow rate, h is height of the bath, and w is width of the bath.For the closed bath chamber, we have Q = 3.6 mL/min = 0.06 cm 3 /s, ղ of PBS = approximately 1.00 × 10 -3 Pa•s, 6 h = 0.25 cm, and w = 1.3 cm (Warner Instruments).Thus, the fluid shear stress is calculated as Converting from Pa to dyn/cm 2 (1 Pa = 10 dyn/cm 2 ), we have an estimated FSS of 0.044 dyn/cm 2 in the chamber under these conditions.

Rates of oxygen consumption: Assumptions and calculations
At each time point, the fluorescence intensities from the oxygen sensor film were collected in mean grey value, which was converted to [dissolved O2] (mM) by using the oxygen calibration curve for the perfusion chamber (Stern-Volmer equation, Eq. 1 from main text) and Henry's law.After generating a plot of [O2] (mM) vs time (s), the initial portion of the curve was fit with a linear fit, and the mean rate of consumption per tissue slice (mM/s) was obtained from the absolute value of the slope (Figure 4d-f).
To compare the optical assay to the results from a Seahorse assay, it was necessary to convert between units of mM/s per T cell zone and pmol/min/cell.Doing so required a series of assumptions and simplifications, yielding an order-of-magnitude comparison between the two measurements.To convert from molarity to moles by using the molarity equation (Eq.S 4), Molarity = moles of substrate / volume of solution Eq. S 4 We estimated the average volume (area x height) of the T cell zone in a lymph node slice.To determine an average area, we performed immunofluorescence labelling of lymph node slices, defined the T cell zones as central B220-negative regions (see e.g. Figure 3c from main text), and measured the areas of these regions using ImageJ.Measurements in 12 naïve lymph node slices from 6 wks old male and female mice yield an average area of 1.01× 10 6 ± 0.26 × 10 6 µm 2 .Assuming a thickness of 300 µm, this yielded an average volume of 3.02 × 10 8 µm 3 .
To convert from moles per T cell zone to moles per cell, we estimated an average number of T cells per T cell zone.To do so, we referred to the average percent composition of CD3+ T cells per lymph node (50 ± 17%) and the average number of cells per 300-µm-thick lymph node slice ((0.56 ± 0.16) × 10 6 cells). 7suming each murine lymph node slice had two T cell zones (most have one or two zones; see Figure S2), this yielded a rough order-of-magnitude estimate of 140,000 T cells per T cell zone in a lymph node slice.
As an example, using these numbers, for a T cell zone in which the oxygen consumption rate was 19 × 10 -4 mM/sec as measured by the optical assay, we estimated that 2.4 × 10 -4 pmol/min/cell was consumed.

Figure S1 :
Figure S1: Characterization of the photostability and absorption and emission spectra of the PdTFPP

Figure S2 :
Figure S2: Distribution of oxygen consumption in lymph node slices from naïve mice.Each image was