Isotopic tracing of glucose metabolites in human monocytes to assess changes in inflammatory conditions

Summary Differences in metabolic profiles can link to functional changes of immune cells in disease conditions. Here, we detail a protocol for the detection and quantitation of 19 metabolites in one analytical run. We provide the parameters for chromatographic separation and mass spectrometric analysis of isotopically labeled and unlabeled metabolites. We include steps for incubation and sample preparation of PBMCs and monocytes. This protocol overcomes the chromatographic challenges caused by the chelating properties of some metabolites.


Ammonium acetate (CH 3 COONH 4 ) buffer stock solution
Timing: 15 min 6. Prepare a 100 mM solution of CH 3 COONH 4 in H 2 O. To obtain 0.5 L of buffer stock solution weigh 3.85 g of CH 3 COONH 4 and bring to volume in a 0.5 L volumetric flask. 7. Adjust pH with ammonia solution (NH 3 ) to pH 9. CRITICAL: NH 3 causes severe skin burns and eye damage. Always wear gloves, google, and lab coat while handing it. It may cause respiratory irritation. Work under fume hood. It may be corrosive to metals, avoid contact. It is very toxic to aquatic life and with long lasting effects. Avoid release to the environment.

MATERIALS AND EQUIPMENT LC-MS setting
For this protocol an Agilent 1290 Infinity II HPLC system was hyphenated to an Agilent 6495 QqQ mass spectrometer (MS) with an Agilent jet stream source with electrospray ionization (AJS-ESI), both controlled by MassHunter Data Acquisition software (Agilent, Waldbronn, Germany). For the separation of the metabolites, an Agilent InfinityLab Poroshell 120 HILIC-Z column (PEEK-lined, 2.1 3 100 mm, 2.7 mm) was used. Table 1 shows the HPLC conditions and Table 2 the MS parameters. Fragmentation and source parameters were optimized using Agilent Optimizer and Agilent Source Optimizer software. The acquisition was conducted in dynamic multiple reaction monitoring (dMRM) mode in both, positive and negative mode.

STEP-BY-STEP METHOD DETAILS
This protocol can be applied to different cell cultures. Conditions of cell incubation will need previous evaluation and adjustment.
We show here, the protocols used for the incubation of PBMCs and monocytes.
Two different conditions were used in both cases: with labeled (1,2-13 C 2 -D-glucose) and unlabeled glucose.

PBMC incubation
Timing: 8-9 h This part describes experimental steps starting with about 40 million PBMCs.
1. Thawing of PBMCs and preparation for the incubation. a. Warm 10 mL washing medium (10% FBS in Roswell Park Memorial Institute (RPMI) 1640 medium) in a falcon tube to 37 C in a water bath. b. Warm 5 mL washing medium containing benzonase (25 U/mL) at 37 C in a water bath. c. Thaw frozen PBMCs (max of 40 3 10 6 cells) in a water bath (37 C). When almost completely thawed, transfer the cells under sterile condition to the falcon tube containing 10 mL washing medium (a., without benzonase). d. Centrifuge at 300 3 g for 10 min at room temperature, then remove the supernatant. e. Gently resuspend each cell pellet in 1 mL of warmed medium with benzonase (b.), then add another 4 mL of benzonase medium. Mix well and incubate at 37 C in a water bath for 5 min. f. Centrifuge at 300 3 g for 10 min at room temperature, then remove the supernatant. 2. PBMC incubation in an ultra-low attachment 6-well plate.
a. Sterile-filter (with 0.2 mm filter) the medium supplemented with either unlabeled or 1,2-13 C 2 -D-glucose (see the paragraph ''before you begin'' points 1.-2. or 3.-4). b. Warm the culture medium (2.a.) to 37 C in a water bath. c. Gently resuspend each cell pellet (1.f.) in the sterilized, warm medium (2.b.) and adjust the cell concentration to 1 3 10 6 /100 mL. d. Transfer about 5 3 10 6 cells (about 500 mL) into an ultra-low attachment surface 6-well plate, add culture medium to a final volume of 1,800 mL. e. Incubate for 2 h at 37 C, 5% CO 2 . f. Add 200 mL of PBS (negative control) or 200 mL of lipopolysaccharide (LPS) solution (100 ng/mL, as a stimulant). The final volume is 2,000 mL/well. g. Incubate at 37 C, 5% CO 2 for another 4 h. 3. Cell harvest. a. Transfer cell suspension into 2 mL Eppendorf tubes. b. Centrifuge at 300 3 g, for 10 min at 4 C. c. Transfer the supernatant into new tubes, then centrifuge at 15,000 3 g, for 10 min at 4 C. Take out 1 mL of supernatant and store at À80 C until measurement. d. Shock freeze the cell pellet in liquid N 2 and leave it for 5 min. e. Take out frozen cell pellet from liquid N 2 , then add 100 mL of H 2 O:ACN (1:1). Vortex thoroughly and incubate on ice for 5 min. f. Centrifuge at 15,000 3 g, for 10 min at 4 C. g. Carefully take 75 mL of the supernatant, without disturbing the cell pellet. Store the cell lysate at À80 C.
CRITICAL: All cell culture experiments should be carried out under laminar flow hood under a sterile condition.
CRITICAL: The use of human cells for research purposes underlies to ethical restrictions. It is necessary to obtain appropriate approvals before starting the research.
CRITICAL: Incubation time should be validated prior to experiment (i.e., the incubation time in 2.e. and g. can be varied and tested).
Note: After isolation, PBMCs were stored in liquid N 2 until the experiment.
Optional: In step 2.f other stimulants may be applied instead of LPS.

Monocyte incubation
Timing: 9-10 h This step begins with about 40 million PBMCs.
4. Thawing of PBMCs. a. Warm 10 mL of medium (10% FBS in RPMI 1640 medium) in a falcon tube to 37 C in a water bath. b. Warm 5 mL of washing medium containing benzonase (25 U/mL) at 37 C in a water bath. c. Thaw frozen PBMCs (max of 40 3 10 6 cells) in a water bath (37 C). When almost completely thawed, transfer the cells under sterile condition to the falcon tube containing 10 mL washing medium (a., without benzonase). d. Centrifuge at 300 3 g for 10 min at room temperature, then remove the supernatant. e. Gently resuspend each cell pellet in 1 mL of warmed medium with benzonase (b.), then add another 4 mL of benzonase medium. Mix well and incubate at 37 C in a water bath for 5 min. f. Centrifuge at 300 3 g for 10 min at room temperature, then remove the supernatant. 5. Separation of monocytes with the magnetic-activated cell sorting (MACS) (negative selection approach using Pan Monocyte Isolation Kit, human). a. Prior to MACS sorting, put the LS column at À20 C, to minimize unspecific binding. b. Wash the cell pellet (4.f.) with 1 mL of MACS buffer (0.5% BSA in PBS containing 2 mM EDTA) and transfer to 1.5 mL Eppendorf tubes. c. Centrifuge at 300 3 g, for 10 min at 4 C, then take out the supernatant. d. Resuspend the cell pellet in 400 mL of MACS buffer (for 5 3 10 6 cells). e. Add 100 mL of FcR blocking reagent (for 5 3 10 6 cells). f. Add 100 mL of biotin-antibody cocktail (for 5 3 10 6 cells). g. Mix well and incubate for 5 min in the refrigerator (2 C-8 C). h. Add 300 mL of MACS buffer (for 5 3 10 6 ).

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i. Add 200 mL of anti-biotin micro beads (for 5 3 10 6 cells). j. Mix well and incubate for 10 min in the refrigerator (2 C-8 C). k. Wash with 1 mL of MACS buffer. l. Centrifuge at 300 3 g, for 10 min at 4 C, take out the supernatant, and then resuspend the cell pellet with 500 mL MACS buffer. m. Place the LS column in the magnetic field of a MACS separator. n. Precondition the column by rinsing with 3 mL of MACS buffer. o. Load the cell suspension (l.) onto the column through the pre-separation filter. p. Collect flow-through, which contains unlabeled cells, representing the enriched panmonocytes. q. Wash column with 3 3 3 mL of MACS buffer and combine all four flow-through. r. Take an aliquot of 10 mL for cell count, then centrifuge the remaining cells at 300 3 g, for 10 min at 4 C and remove supernatant. 6. Monocytes incubation in an ultra-low attachment 24-well plate.
a. Sterile-filter (with 0.2 mm filter) the medium supplemented with either unlabeled or 1,2-13 C 2 -D-glucose (see the paragraph ''before you begin''). b. Warm the culture medium to 37 C in a water bath. c. Gently resuspend each cell pellet (5.r.) in the sterilized, warm medium (6.b.) and adjust the cell concentration to ca. 8 3 10 5 /100 mL. d. Transfer about 8 3 10 5 cells (about 100 mL) of cell suspension into an ultra-low attachment surface 24Àwell plate and add culture medium to a final volume of 300 mL. e. Add 0.3 mL of Monensin per well. f. Incubate for 5 h at 37 C, 5% CO 2 . 7. Cell harvest.
a. Transfer cell suspension in 2 mL Eppendorf tubes. b. Centrifuge at 300 3 g, for 10 min at 4 C. c. Separate the supernatant from the cell pellet (attention not to disturb cell pellet: do not aliquot the entire volume of supernatant). To analyze the culture medium, centrifuge it at 15,000 3 g, for 10 min at 4 C before LC-MS analysis. d. Shock freeze the cell pellet into liquid N 2 and leave it for 5 min. e. Take out from liquid N 2 and add 100 mL of H 2 O:ACN (1:1). f. Vortex thoroughly and incubate on ice for 5 min. g. Centrifuge at 15,000 3 g, for 10 min at 4 C. h. Carefully take 75 mL of the supernatant, without disturbing the cell pellet to obtain the samples of cell extract. i. Put the samples at À80 C or on dry ice.
CRITICAL: The incubation of monocytes is particularly delicate in a culture medium without pyruvate and glutamine. Verify regularly during the incubation the well-being of the cells and consider that reaching the isotopic steady state might be challenging.
CRITICAL: All cell culture experiments should be carried out under laminar flow box in a sterile environment.
CRITICAL: The use of human cells for research purposes underlies to ethical restrictions. It is necessary to obtain appropriate approvals before starting the research.
Note: After collection and before the incubation, PBMCs from where monocytes were extracted, were stored at À80 C.
Optional: In step 6.e different stimulants can be used, for instance, LPS to simulate different incubation conditions.

Preparation of mobile phases
Timing: 15 h 8. Deactivation solution A (mobile phase A: 10 mM CH 3 COONH 4 in H 2 O + InfinityLab deactivator additive). a. To obtain 1 L of mobile phase A, add 100 mL of CH 3 COONH 4 stock solution (''before you begin'') to 900 mL of milli-Q water. b. Add 1 mL of InfinityLab deactivator additive per liter of mobile phase (final concentration of 5 mM). c. Let it rest overnight at room temperature. d. Filter with a 2 mm filter (non-sterile, nylon, 0.2 mm, 47 mm). e. Sonicate the mobile phase for 5-10 min to degas. 9. Deactivation solution B (mobile phase B: 10 mM CH 3 COONH 4 in ACN + InfinityLab deactivator additive). a. To obtain 1 L of mobile phase B, add 100 mL of CH 3 COONH 4 stock solution (''before you begin'') to 900 mL of LC-MS grade ACN. b. Add 1 mL of InfinityLab deactivator additive per liter of mobile phase (final concentration of 5 mM). c. Let it rest overnight at room temperature. d. Filter with a 2 mm filter (non-sterile, nylon, 0.2 mm, 47 mm). e. Sonicate the mobile phase for 5-10 min to degas.
CRITICAL: There might be some precipitation in the mobile phases, especially in the organic one (B). It is recommended to add the buffer stock solution slowly to the ACN, and only after 10-15 min the InfinityLab deactivator additive.
CRITICAL: ACN is toxic by oral ingestion, dermal contact, and inhalation. It also causes eye irritation. Always use gloves, google, and lab coat and work under fume hood while handling it.
Passivation and conditioning of the system Timing: 18-19 h The passivation and conditioning of the system was conducted accordingly to Agilent's protocol for the use of the InfinityLab deactivator (Agilent Technologies, 2018).

Phosphoric acid wash.
a. Put milli-Q water as mobile phase for both channels. b. Purge the system for 5 min at 5 mL/min directly to waste. If the system does not have a purge valve, momentarily detach the column, and put the inlet capillary to a waste container. c. Set the flow of milli-Q water to 0.25 mL/min and run for 30 min through the system and the column. d. Change the flow rate to 0 mL/min. e. Take out the spray needle from the MS source and fix it vertically in a waste container (Figure 1). Do not inject phosphoric acid wash in the MS. f. Switch the solvent in both channels to the 0.5% phosphoric acid wash (''before you begin: passivation solution: 0.5% phosphoric acid wash''). g. Purge the system, for 5 min at 5 mL/min with the phosphoric acid wash. h. Set the flow of 0.5% phosphoric acid wash to 0.1 mL/min and run for 14 h. i. Change the flow rate to 0 mL/min. j. Switch the solvent in both channels to milli-Q water. ll OPEN ACCESS k. Purge the system at 5 mL/min for 10 min with milli-Q water. l. Set the flow of milli-Q water to 0.25 mL/min and run for 1 h through the system and the column. m. Change the flow rate to 0 mL/min. n. Switch the solvent to mobile phase A and B (''mobile phases preparation''). o. Purge the system with mobile phases A and B (50:50) at 5 mL/min for 5 min. p. Reinstall the spray needle into the MS.
CRITICAL: Take out the spray needle from the MS during the phosphoric acid wash. Do not inject phosphoric acid into the MS.
CRITICAL: During the passivation keep the spray needle in a vertical position, as shown in Figure 1, and let the sheath gas flow to prevent the formation of persistent drops of phosphoric wash along the capillary.
a. Set the flow of the mobile phase to 0.2 mL/min (60% A -40% B) and run for 30 min through the system and column. b. Set the flow of the mobile phase to 0.3 mL/min (60% A -40% B) and run for 15 min through the system and column. c. Change the composition to 50% A -50% B and run for 30 min through the system and column. d. Change the composition to 10% A -90% B and run for at least 1 h through the system and column.
CRITICAL: The step-by-step increase of the percentage of mobile phase B, minimizes the risk of precipitate formation in the system.

HPLC-MS analysis
Timing: 21 min per run 12. After conditioning of the analytical column, it is possible to start the analysis.   Table 3 shows the details of the dMRM method.
CRITICAL: Always run a couple of blanks before starting the analysis to be sure that the column is well conditioned and the pressure stable. Be aware that analysis with HILIC needs longer column conditioning.
CRITICAL: There might be some precipitation in the mobile phases. To our knowledge there is no suitable pre-column for both conditions of phosphoric wash and pH 9 analysis, thus the use of an in-line filter is recommended to preserve the column.
Troubleshooting 1/ Problem 1: Precipitation in the mobile phases.
If precipitation occurs in the mobile phases (particularly in B) there will be some drops in the pressure curve of the instrument. See the protocol section ''troubleshooting, problem 1'' for more details.

EXPECTED OUTCOMES
Data were obtained from the incubation of PBMCs and monocytes. Figure 2 shows the metabolic pathways considered, and the intermediate metabolites highlighted in red were identifiable and quantifiable in this study. The incubation of 5 million PBMCs was conducted in two different conditions: with unlabeled glucose and 1,2-13 C 2 labeled glucose. The amounts of the above-mentioned metabolites after incubation with glucose are shown in Figure 3 (unlabeled glucose) and Figure 4 (labeled glucose). As result, most of the labeled glucose was transformed into lactate and barely reached the TCA cycle. As discussed before, the labeled glucose may require several hours to reach the TCA cycle.
Monocytes (N=8 3 10 5 ) were incubated in two different conditions: without a stimulation, therefore only with medium (CON) and with the addition of lipopolysaccharides (LPS). Both, cell extract and medium of the incubation, were analyzed.
The presence of lactate, glutamine, and amino acids in the incubation medium is not unexpected. On the contrary, the detection of glutamic acid, pyruvate, and citric acid is a warning sign of the well-being of the cells. These compounds cannot pass through the cell membrane, and therefore, their presence in the medium is probably due to the disruption of the membrane after the death of the cells.
In conclusion, this protocol allowed the detection and quantitation of specific compounds that are necessary to have a general overview of the well-being or the metabolic alterations of the cells. Depending on the focus of the future research and on the typology of cells used, the protocol might need adaptations. We gave an example of application to the analysis of PBMCs and monocytes, highlighting pros and cons of the method.

Figure 2. Metabolic pathways considered in this study
The compounds highlighted in red were detectable and quantifiable.

QUANTIFICATION AND STATISTICAL ANALYSIS
The method was validated based on the ICH guideline M10 on bioanalytical method validation (EMA, 2019).
Since the matrix used is rare (PBMCs and monocytes), the validation was performed in double blanks (ACN:H 2 O), except for the matrix effect and the recovery study. Therefore, stock solutions and
Some of the targeted analytes, such as 1,2-13 C 2 lactate, 2,3-13 C 2 serine, 1 13 C acetylCoA, 1-13 C ribose-5-phosphate, 2,3-13 C 2 glyceraldehyde, 1,2-13 C 2 fructose-6-phosphate, 1,2 13 C 2 phosphoglyceric acid, 1À 13 C ATP, and 1,2-13 C 2 citric acid are not commercially available to our knowledge. It is assumed, that the retention times of the labeled compounds are the same of the corresponding  unlabeled ones, allowing for the identification of the targeted analyte. The suitable transitions were hypothesized based on fragmentation patterns of the unlabeled analytes. For some of them (lactate, fructose-6-phosphate, phosphoglyceric acid, ATP) the fragmentation patterns were confirmed by the results of the cell extract of PBMCs. Figure 9 shows the general chromatogram of the unlabeled substances. The retention times of the relative labeled substances are virtually the same. Those analytes that could not be chromatographically separated could be distinguished by different MRM transitions as shown in Table  3.
Some compounds present in the matrix show the same molecular weight and the same ion transitions and, therefore, cause interference in the identification and quantitation. This is the case for glucose-6-phosphate and fructose-6-phosphate that have the same precursor and the same product ions and the transitions are listed in Table 3. As shown in Figure 10 though, they are chromatographically separated.

Figure 7. Concentration of unlabeled compounds in the incubation medium
The incubation was performed with 8 3 10 5 monocytes for 5 h with the addition of 1,2-13 C 2 glucose to the medium. The incubation was conducted in two different conditions: without stimulation (CON) and with the addition of LPS (LPS). The experiments were conducted in triplicates. All data points are illustrated in the graphic, and the bars represent the mean value G SD.

Figure 8. Concentrations of labeled compounds in the incubation medium
The incubation was performed with 8 3 10 5 monocytes for 5 h with the addition of 1,2-13 C 2 glucose to the medium. The incubation was conducted in two different conditions: without stimulation (CON), with the addition of LPS (LPS).The experiments were conducted in triplicates. All data points are illustrated in the graphic, and the bars represent the mean value G SD.

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Citric acid and isocitrate have the same fragmentation pattern except for the transition m/z 191.0 / 73.0 which is characteristic for the isocitrate only. Unfortunately, they are not chromatographically separable.

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Glyceraldehyde-3-phosphate presents two chromatographic peaks (Figure 11), probably due to the conversion to the enolic form as shown by the structures in Figure 12.
Glyceraldehyde and dihydroxyacetone phosphate (DHAP) have the same molecular weight and fragmentation pattern (m/z 169 / 79.1, m/z 169 / 96.9) but they are chromatographically separated as shown in Figure 13.

Calibration curve
For the calibration curves, at least 8 concentration levels of calibration standards were used, including lower limit quality control (LLQC), lower limit of quantitation (LOQ), middle quality control (MQC), and high quality control (HQC). The Mandel test was performed to assess the better fitting, linear or quadratic, the analysis of the variances was conducted, and the values of LOD and LOQ were calculated with the intercept of the linear regression (LOD= 3.3 3 standard error intercept/slope; LOQ= 103 standard error intercept/slope). Table 4 summarizes the regression data, the LOD, and the LLOQ.

Matrix effect
In electrospray ionization, matrix effect is a confounding factor that may have a strong impact on the peak areas due to variations of ionization yield of the individual analyte.
The matrix effect was evaluated for all the target analytes in PBMCs at three different concentrations: high, medium, and different low concentrations. The analytes are endogenous compounds; therefore, their amounts were evaluated before (blank matrix) and after the spike (spiked matrix) at high, matrix effect ð%Þ = spiked matrix À blank matrix spiked double blank 3 100 Results are shown in Figure 14 for the unlabeled compounds and in Figure 15 for the labeled compounds.

Recovery
The recovery was evaluated for all the target analytes in PBMCs at three different concentrations: 10 mg/mL (HIGH), 5 mg/mL (MEDIUM), and different low concentrations (LOW): 0.01 mg/mL for acetyl CoA, 13 C AMP, ATP; 13 C glutamic acid, 0.05 mg/mL for 13 C glutamine, 13 C glycine, 0.1 mg/mL for glutamic acid and glutamine, 0.25 mg/mL for pyruvate and 13 C pyruvate, 0.5 mg/mL for citric acid, fructose-6-phosphate, glyceraldehyde-3-phosphate, glycine, phosphoglyceric acid, 1 mg/mL for ribose-5-phosphate, serine, and lactate. The results obtained are shown in Figure 16 for the unlabeled compounds and in Figure 17 for the labeled compounds.

Accuracy and precision
Intra-day and inter-day precision and accuracy were evaluated for all compounds. Four concentrations (LLQC, LQC, MQC, HQC) were injected in quintuplicates three times on the same day (intra day) and on three different days (inter-day). The results were within G15% for CV% (precision) and G15% for RE% for all the concentration levels. Details are reported in Table 5.   The standards were spiked at three different concentrations, high (10 mg/mL), medium (5 mg/mL), and low: 0.01 mg/mL for acetyl CoA, ATP, 0.05 mg/mL for AMP, 0.1 mg/mL for glutamic acid and glutamine, 0.25 mg/mL for pyruvate, 0.5 mg/mL for citric acid, fructose-6-phosphate, glyceraldehyde-3-phosphate, glycine, phosphoglyceric acid, 1 mg/mL for ribose-5-phosphate, serine, and lactate. All measurements were conducted in sextuplicate. Data are represented as mean G SD.
Moreover, the freeze-thaw stability was evaluated after three cycles. The obtained results are all within G15%.

LIMITATIONS
A limitation of this method is the number of cells that are necessary to obtain a sufficient concentration of compounds to analyze and quantify. Therefore, is not recommended to apply this protocol to experiment sets with a very limited number of cells.
The concentrations taken into consideration in this protocol are wide, and there is a high variability of concentrations in the cell extract. For instance, the concentrations of lactate are clearly in a different range in comparison with ATP or AMP. The standards were spiked at three different concentrations, high (10 mg/mL), medium (5 mg/mL), and low: 0.01 mg/mL for 1-13 C AMP, and 1,2-13 C glutamic acid, 0.05 mg/mL for 1,2-13 C 2 glutamine, and 2À 13 C glycine, 0.25 mg/mL 2,3-13 C 2 pyruvate. All measurements were conducted in sextuplicate. Data are represented as mean G SD. Figure 16. Recovery studies for the targeted unlabeled compounds The standards were spiked at three different concentrations, high (10 mg/mL), medium (5 mg/mL), and low: 0.01 mg/mL for acetyl CoA, ATP, 0.05 mg/mL for AMP, 0.1 mg/mL for glutamic acid and glutamine, 0.25 mg/mL for pyruvate, 0.5 mg/mL for citric acid, fructose-6-phosphate, glyceraldehyde-3-phosphate, glycine, phosphoglyceric acid, 1 mg/mL for ribose-5-phosphate, serine, and lactate. All measurements were conducted in sextuplicate. Data are represented as mean G SD.

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This protocol describes an in vitro study; therefore, it is a model that might differ from the metabolism in vivo. This aspect must be taken into consideration while evaluating the data.

TROUBLESHOOTING
Problem 1 Precipitation in the mobile phases.
After some days, if the temperature of the laboratory is not highly controlled, the formation of precipitate in the mobile phases may be observed. As a result, the pressure of the system will show a general increase and some quick and temporary pressure drops as shown in Figure 18. After a while, these drops will become more frequent and longer.
Since there is not a pre-column capable to resist both pH, acidic for the passivation and basic for the analysis, the introduction of an in-line filter with a pore size of 0.2 mm or 0.3 mm is recommended to prevent the possible occlusion of the column due to precipitation in the mobile phases.

Potential solution
The best way to solve these pressure drops is to clean the system with pure water for 15-30 min and redo the passivation of the system afterwards.

Problem 2
Citric acid peak is difficult to integrate.

Potential solution
It is extremely important to use as much as possible steel-free capillaries, column fittings, and connectors. Another possible solution, not yet tested, is the use of a bioinert system.

Problem 3
Some cells, like monocytes, show some vulnerability during the incubation in a medium without glutamine and pyruvate and start to die already after 4-5 h.

Potential solution
Do not plan very long incubation of these vulnerable cells or optimize the incubation conditions in advance. Remember also that the incorporation of labeled glucose into the TCA cycle takes a longer time, and the use of labeled glutamine instead might be considered. The standards were spiked at three different concentrations, high (10 mg/mL), medium (5 mg/mL), and low: 1-13 C AMP; 1,2-13 C 2 glutamic acid, 0.05 mg/mL for 1,2-13 C 2 glutamine, 2-13 C glycine, 0.25 mg/mL 2,3-13 C 2 pyruvate. All measurements were conducted in sextuplicate. Data are represented as mean G SD. Problem 4 Some chromatographic peaks, like citric acid, might need manual integration to obtain the correct quantitation.

Potential solution
Carefully review every data and integrate manually, if necessary. Be consistent, in calibration curves and samples, to obtain a correct quantitation.

Problem 5
If PBMCs are obtained from blood donations, they might not be checked for infectious diseases.

Potential solution
Be sure to handle the samples with the right laboratory equipment: always wear gloves, lab coat, and googles.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Maria K. Parr (maria.parr@fu-berlin.de).

Materials availability
This study did not generate new unique reagents.

Data and code availability
This study did not generate original code.

ACKNOWLEDGMENTS
Ginevra Giacomello received a scholarship from the NeuroMac School (DFG, the German Research Foundation -Project-ID 259373024 -CRC/TRR 167 (B05)) and was funded by the State of Berlin, Germany, with the Elsa-Neumann PhD scholarship. We would like to acknowledge the support by the OpenAccess Publication Fund of Freie Universitä t Berlin and the assistance of the Core Facility BioSupraMol supported by the DFG. We thank Bernhard Wü st, Agilent Technologies Inc., for his precious assistance in mass spectrometry and Adeline Dehlinger and Christian Bö ttcher for the sample collection of CSF and PBMCs and the introduction of G.G. to cell culture.