*2.3. XF96 Extracellular Flux Analysis in Fresh and Frozen Cells*

In all respirometry assays, lymphocytes and LCLs were spun down on poly-d-lysinecoated plates on the day of the assay. LCLs were plated at 200,000 cells per well and lymphocytes were plated at 400,000 cells/well. Cells were added to the plate at a total volume of 175 µL per well and centrifuged at 400× *g* for 5 min with no break. Table 2 presents the mitochondrial-targeted compounds and substrates used and the associated mechanism of action.

#### 2.3.1. Intact Cell Respirometry

Intact lymphocytes respirometry was measured in a DMEM assay medium containing 5 mM glucose, 2 mM glutamine, 1 mM pyruvate, and 5 mM HEPES. Injections resulted in a final concentration of 2 µM oligomycin from Port A, 3 µM FCCP from Ports B and C, and 2 µM antimycin A and rotenone from Port D. Measurements included three basal measurements, three measurements after injection of oligomycin, four FCCP measurements, and three antimycin A and rotenone measurements. Respiration measurements were conducted with four technical replicates.

**Table 2.** The mitochondrial compounds/substrates used to investigate mitochondrial function in fresh and frozen samples. The compounds used to measure mitochondrial respiratory function and their corresponding functions are listed. Abbreviations: ADP: Adenosine diphosphate; ATP: Adenosine triphosphate; CI: Complex I; CII: Complex II; CIII: Complex III; CIV: Complex IV; CV: Complex V; ETC: Electron transport chain; NADH: Reduced nicotinamide adenine dinucleotide; OXPHOS: Oxidative phosphorylation; TMPD: N, N, N', N'-tetramethyl-p-phenylenediamine.


#### 2.3.2. Permeabilized Cell Respirometry

Permeabilized lymphocytes respirometry was measured in Mitochondrial Assay Solution (MAS) (70 mM Sucrose, 220 mM Mannitol, 5 mM KH2PO4, 5mM MgCl2, 1 mM EGTA, 2 mM HEPES; pH 7.2) containing 4 mM ADP, 2 µM rotenone, 5 mM succinate, and 5 nM XF PMP. Cells were spun down in Seahorse assay medium and then washed twice with MAS before adding MAS supplemented with ADP, succinate, rotenone, and XF PMP. Cells were incubated at 37 ◦C for 10 min before initiating the respirometry assay with the permeabilized cells starting in State 3 respiration. Injections resulted in a final concentration of 2 µM oligomycin from Port A, 1 µM FCCP from Port B, and 2 µM antimycin A and rotenone from Port C, and 0.5 mM TMPD and 1mM Asc from Port D. Measurements included three State 3 measurements, three measurements after injection of oligomycin, two FCCP measurements, two antimycin A and rotenone measurements, and three measurements after injection of TMPD/Asc. Respiration measurements were conducted with four technical replicates.

#### 2.3.3. RIFS

Respirometry in previously frozen lymphocytes was measured in MAS. Cells were resuspended and spun down in 20 µL of unsupplemented MAS. The samples were centrifuged at 2100× *g* for 10 min and stopped without a break. The samples were brought to a final volume of 150 µL with MAS supplemented with 10 µg/mL cytochrome c and 2.5 µg/mL alamethicin. Injections resulted in a final concentration of either 2 µM rotenone and 5 mM succinate or 1 mM NADH from Port A, 2 µM antimycin A and rotenone from Port B, 0.5 mM TMPD and 1mM Asc from Port C, and 50 mM sodium azide from Port D. Measurements included one measurement before injection of substrates (NADH or succinate and rotenone), two maximal respiration measurements after injection of substrates, two measurements after injection of antimycin A and rotenone, two TMPD/Asc measurements, and two measurements after inhibition of CIV with azide. Respiration was measured in triplicate.

#### 2.3.4. Complex V ATP Hydrolysis Assay

ATP hydrolysis was measured in frozen samples in MAS. Cells were resuspended and spun down in unsupplemented MAS (20 µL of cell suspension). After spinning the cells in the XF96 plate 130 µL MAS containing 5 mM succinate plus 2 µM rotenone was added per well. Injections resulted in a final concentration of 2 µM antimycin A from Port A, 10 mM ATP plus 1 µM FCCP from Port B and C. Port B and C are injected consecutively to assess maximal ATP concentration. Measurements included two measurements before injection of Port A, two measurements after injection of Port A and three maximal ATP hydrolytic activity (ECAR) after injection of Port B/C. Respiration was measured in triplicate.

#### *2.4. MitoTracker Deep Red in Frozen Cells*

Cells were stained with 500 nM MTDR in PBS for 10 min in a black 96-well plate imaging plate. Cells were then washed with PBS to remove the MTDR. Fluorescence intensity was measured from the bottom of the plate in a Tecan Spark 10 M Multimode Plate Reader using an excitation wavelength of 633 nm and an emission wavelength of 678 nm.

#### *2.5. Enzymatic Assays in Frozen Cells*

#### 2.5.1. Complex I Activity

CI activity was measured in cell lysates following NADH oxidation at 340 nm as described previously [25].

#### 2.5.2. Citrate Synthase Activity

Citrate synthase activity was performed in cell lysates using DTNB, as described in [5].

#### *2.6. Statistical Analysis*

Assays were run as proof of concept that the RIFS protocol is comparable to traditional CI spectrophotometric assays and only run in one independent experiment. Therefore, statistics were not performed on the data.

#### **3. Gold Standards: Human Samples for Assessment of Mitochondrial Bioenergetic Function**
