The authors would like to thank the animal facility of the Max Planck Institute of Immunobiology and Epigenetics for providing the animals used in this study.

Breeding and husbandry of all mice used for this protocol were conducted in a conventional animal facility at the Max Planck Institute for Immunobiology and Epigenetics (MPI-IE) according to the regulations of the local authorities (Regierungspr&#228;sidium Freiburg). Mice were euthanized with CO2 and cervical dislocation by FELASA B-trained personnel following guidelines and regulations approved by the animal welfare committee of the MPI-IE and the local authorities. No animal experimentation was performed, and mice were without health burdens.
NOTE: Highly sensitive analytical methods such as the LC-MS method used in this protocol have the potential to detect even very minor contaminations in the sample. Therefore, it is of utmost importance to minimize such contaminations. The single most important measure is to wear clean gloves whenever touching anything that comes in contact with the samples. This includes for example, preparation of buffers and sheath fluid, labeling of sample tubes, and cleaning laboratory equipment. Moreover, single-use labware should be used whenever possible. Glassware should be rinsed with LC-MS grade solvents prior to use. A clean working environment further helps reduce contaminations.
1. General preparations
<ol>
	<li>Prepare internal standard stock solution: Prepare 6 mg/mL chloro-phenylalanine (CLF) and 6 mg/mL aminoterephtalic acid (ATA) in 30% v/v 2-propanol in ultrapure water.</li>
	<li>Prepare FACS resuspension buffer: Add 0.9 g of NaCl, 99.5 mL of ultrapure water, and 0.5 mL of internal standard stock solution. Pre-cool to 4 &#176;C.</li>
	<li>Prepare sheath fluid: In a sheath fluid tank, dissolve 30 g of NaCl in 6 L of deionized water and connect to a cell sorter. 
	&#8203;NOTE: The concentration of NaCl in the sheath fluid has been optimized to allow deflection of droplets in the sorter that is as robust as when using PBS as sheath fluid and simultaneously to minimize negative effects on metabolite detection by LC-MS12. Lower concentrations of NaCl are advantageous for metabolite detection but impair the robust sorting of cells.</li>
</ol>
2. Isolation of B and T cells from the spleen
<ol>
	<li>Pre-cool 90 mL of phosphate-buffered saline (PBS) per mouse in a fridge or on ice to 4 &#176;C. Pre-cool a centrifuge to 4 &#176;C.</li>
	<li>Isolation of spleens 
	NOTE: It is most critical to work fast, on ice, and with cold (4 &#176;C) buffers during cell isolation and the following staining steps. Otherwise, the metabolic profile of cells might change considerably.
	<ol>
		<li>Prepare 1.5 mL of PBS in a 2 mL reaction tube on ice.</li>
		<li>Euthanize a C57BL6/J mouse according to the guidelines and methods approved by local authorities.</li>
		<li>Sterilize fur with 70% ethanol.</li>
		<li>Open the abdomen with scissors and remove the spleen using fine forceps without rupturing the organ. Immediately place the spleen in cold PBS (4 &#176;C).</li>
	</ol>
	</li>
	<li>Generation of a single-cell suspension
	<ol>
		<li>Place a 70 &#181;m cell strainer on a 50 mL centrifuge tube and wet it with PBS. Pass the spleen through the mesh using the thumb rest of a syringe plunger and 30 mL of cold PBS (4 &#176;C)</li>
		<li>Centrifuge at 300 x g for 5 min. Remove the supernatant.</li>
		<li>Resuspend the pellet in 1 mL of ACK lysis buffer and incubate for 2 min at room temperature (RT; 20 &#176;C).</li>
		<li>Add 50 mL of cold PBS (4 &#176;C). Centrifuge at 300 x g for 5 min and discard the supernatant.</li>
	</ol>
	</li>
	<li>FACS staining
	<ol>
		<li>Prepare the antibody cocktail in 500 &#181;L of cold PBS (4 &#176;C) per mouse: CD3-PE (1:200), B220-A647 (1:200).</li>
		<li>Resuspend the cell pellet in the antibody cocktail. Transfer to a 5 mL FACS tube. Incubate for 30 min at 4 &#176;C in the dark.</li>
		<li>Add 3 mL of cold PBS (4 &#176;C). Centrifuge at 300 x g for 5 min and discard the supernatant.</li>
		<li>Resuspend the cell pellet in 1 mL of cold FACS resuspension buffer (4 &#176;C). Filter the cells through a filter-cap FACS tube. Cells are ready for flow cytometry-based sorting.</li>
	</ol>
	</li>
</ol>
3. Isolation of HSCs and MPPs from bone marrow
<ol>
	<li>Dissect and isolate the bone marrow. 
	NOTE: It is most critical to work fast and keep isolated bones and the cells cold (4 &#176;C) during the whole procedure to allow accurate results. Furthermore, to minimize background signals, work and use a clean space and laboratory equipment.
	<ol>
		<li>Euthanize mice according to guidelines and methods approved by local authorities.</li>
		<li>Spray 70% Ethanol on the lower part of the mice.</li>
		<li>Dissect the legs and the spines using scissors. Make an incision on the back and extend the cut around to the abdomen. Peel the skin from the hind limbs.</li>
		<li>Rip or cut the hind limbs off, and make sure the legs contain the tibia, femur, and hip joint.</li>
		<li>Take the scissors and cut them close alongside the spine until completely removed.</li>
		<li>Distribute the legs and spines into 6-well-plates containing cold PBS (4 &#176;C) and keep the plates on ice.</li>
		<li>Clean the femur, tibia, hip joint, and vertebrae of soft tissue using a scalpel and scissors.</li>
		<li>Crush the cleaned bones with a mortar and pestle in 5 mL of cold PBS (4 &#176;C).</li>
		<li>Filter the cell suspension through a 40 &#181;m cell strainer into a 50 mL tube.</li>
		<li>Repeat steps 3.1.8-3.1.9&#160;until the bones appear completely white.</li>
	</ol>
	</li>
	<li>Lysis of erythrocytes:
	<ol>
		<li>Centrifuge the harvested cell suspension at 400 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
		<li>Resuspend the cell pellet in 1 mL of cold ACK buffer (4 &#176;C). Incubate for 5 minutes on ice.</li>
		<li>Add cold PBS (4 &#176;C) to stop the reaction (optional).</li>
		<li>Centrifuge at 400 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	</ol>
	</li>
	<li>Lineage depletion: 
	NOTE: To enrich for lineage negative (Lin-) cells, a negative selection kit for CD4 cells was used.
	<ol>
		<li>Prepare the magnetic beads:
		<ol>
			<li>Add 500 &#181;L of beads into 2 mL microcentrifuge tubes. Place the tubes on a magnet and discard the supernatant.</li>
			<li>Wash the beads with 1 mL of cold PBS (4 &#176;C) and discard the supernatant.</li>
			<li>Add 500 &#181;L of cold PBS (4 &#176;C) and keep the beads cold (4 &#176;C).</li>
		</ol>
		</li>
		<li>Incubate the cells with 500 &#181;L of lineage cocktail (50 &#181;L of antibody provided by the kit + 450 &#181;L of PBS) for 25 min (shaking, 4 &#176;C) in the dark.</li>
		<li>Wash the cells with cold PBS (4 &#176;C).</li>
		<li>Centrifuge the tube at 400 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
		<li>Resuspend the cells in 1000 &#181;L of cold PBS (4 &#176;C) and transfer the cell suspension to the bead solution.</li>
		<li>Incubate for 15 min on a rotating wheel at 4 &#176;C.</li>
		<li>Place the tubes on a magnet and wait until the solution clears. Transfer the supernatant to a fresh FACS tube.</li>
		<li>Wash the beads with 500 &#181;L of PBS.</li>
		<li>Place the tubes on a magnet and wait until the solution clears.</li>
		<li>Transfer the supernatant to the fresh FACS tube.</li>
		<li>Centrifuge at 400 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	</ol>
	</li>
	<li>Staining for FACS
	<ol>
		<li>Prepare 300 &#181;L of antibody mix per mouse in cold PBS (4 &#176;C).</li>
		<li>Antibody mix for stem/progenitor cells: HSCs: CD4(1:1000)/ CD8a(1:1000)/ B220(1:1000)/ Ter119(1:500)/ Gr-1(1:1000)/ CD11b - PeCy7(1:1000), c-Kit - PE(1:1000), Sca1 - APC-Cy7(1:500), CD48 - BV421(1:1000), CD150 - BV605(1:300)</li>
		<li>Incubate the cells for 40 min in the dark at 4 &#176;C.</li>
		<li>Wash with 1 mL of cold PBS (4 &#176;C). Centrifuge at 400 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
		<li>Resuspend the cell pellet in 1 mL of FACS resuspension buffer and filter the cells through a filter-cap FACS tube.</li>
	</ol>
	</li>
</ol>
4. FACS Sorting
<ol>
	<li>Set up the cell sorter.
	<ol>
		<li>Insert 70 &#181;m nozzle tip. Activate 405 nm, 488 nm, 561 nm, and 633 nm lasers.</li>
		<li>Set sort mode to 4 Way Purity: yield mask 0, purity mask 32, phase mask 0</li>
		<li>Set the drop frequency to 90,400 Hz, and adjust the amplitude and drop delay according to the manufacturer&#39;s instructions. Set plate voltage to 5000 V.</li>
		<li>Define sorting gates according to the manufacturer&#39;s instructions.
		<ol>
			<li>For all cell types, first, select single cells based on forward scatter and sideward scatter signal, followed by selection based on cell type-specific staining.</li>
			<li>For B cells, select the AF647-positive, PE-negative population.</li>
			<li>For T cells, select the PE-positive, AF647-negative population.</li>
			<li>For HSCs, select the PE-Cy7-low, PE-positive, APC-Cy7-positive, BV605-positive, BV421-negative population.</li>
			<li>For MPPs, select the PE-Cy7-low, PE-positive, APC-Cy7-positive, BV421-positive, BV605-negative, population.</li>
			<li>For debris samples, select events that are too small to represent intact cells based on forward scatter and sideward scatter signal. Typical FACS plots showing these gating strategies are shown in Figure 2 and Figure 3.</li>
		</ol>
		</li>
		<li>Adjust the flow rate to obtain less than 15,000 events/s.</li>
	</ol>
	</li>
	<li>Prepare collection plate
	<ol>
		<li>Prepare yeast extract stock solution: Resuspend 1 aliquot of 13C yeast extract in 7.5 mL of ultrapure H2O and 2.5 mL of methanol.</li>
		<li>Prepare extraction buffer: Add 10 &#181;L of 13C yeast extract stock solution to 10 mL of acetonitrile and mix.</li>
		<li>Prepare collection plate: Add 25 &#181;L of the extraction buffer to each well of a 96 well PCR plate that will be used to collect the sample. To minimize evaporation, cover wells with PCR lids (stripes cut into singlets).</li>
	</ol>
	</li>
	<li>Perform the cell sort.
	<ol>
		<li>Open the collection wells as needed to collect sorted cells and close them as soon as possible to minimize evaporation.</li>
	</ol>
	</li>
	<li>If samples cannot be measured immediately, store them in sealed containers at -80 &#176;C for several days. After thawing, sonicate the samples for 5 min to ensure complete resuspension of metabolites.</li>
</ol>
5. LC-QQQ-MS measurement
<ol>
	<li>Prepare LC Solvents
	<ol>
		<li>Prepare medronic acid stock solution (100 mM): Dissolve 17.6 mg of medronic acid in 1 mL of ultrapure H2O.</li>
		<li>Prepare Buffer A: Dissolve 1.92 g of ammonium carbonate in 1 L of ultrapure H2O, and add 50 &#181;L of medronic acid stock solution.</li>
		<li>Prepare Buffer B: Mix 50 mL of buffer with 450 mL of acetonitrile.</li>
		<li>Prepare LC test mix: Mix 1 ppm each of leucine, isoleucine, AMP, ADP, and ATP in 80:20 acetonitrile: ultrapure H2O. 
		NOTE: Medronic acid stock can be stored at -20 &#176;C for several months; other buffers can be kept at RT for 2 days.</li>
	</ol>
	</li>
	<li>Program LC-QQQ-MS method
	<ol>
		<li>Optimize compound-specific MS settings (e.g., collision energy) according to the manufacturer&#39;s instructions. For Agilent 6495-series instruments, use the settings in Supplementary Table 1.</li>
		<li>Optimize source-specific MS settings according to the manufacturer&#39;s instructions. For instruments with JetStream heated ESI source, use the following parameters: gas temp: 240 &#176;C, gas flow: 15 L/min, nebulizer: 50 psi, sheath gas temp: 400 &#176;C, sheath gas flow: 11 L/min, capillary voltage: 2000 V, nozzle voltage: 300 V.</li>
		<li>Optimize settings of ion transfer optics according to the manufacturer&#39;s instructions. For Agilent instruments with iFunnel ion optics, use the following parameters: high-pressure RF positive: 110 V, high-pressure RF negative: 90 V, low-pressure RF positive: 80 V, low-pressure RF negative: 60 V.</li>
		<li>Program the LC gradient: 0 min, 95% B, 150 &#181;L/min; 18 min, 55% B, 150 &#181;L/min; 19 min, 30% B, 150 &#181;L/min; 21.5 min, 30% B, 150 &#181;L/min; 22 min, 95% B, 150 &#181;L/min; 22.7 min, 95% B, 200 &#181;L/min, 23.5 min, 95% B, 400 &#181;L/min; stop time 28 min. Column temperature: 35 &#176;C.</li>
	</ol>
	</li>
	<li>Set up LC-MS instrument.
	<ol>
		<li>Connect the chromatographic column in the temperature-controlled column compartment.</li>
		<li>Connect the buffers and purge all lines.</li>
		<li>Run at least 4 blank samples or pool samples to equilibrate the column.</li>
	</ol>
	</li>
	<li>Run LC test mix to check chromatographic performance. Ensure the following criteria are met. If not, clean or troubleshoot the instrument before running the samples.
	<ol>
		<li>Confirm that the initial backpressure is &#60;150 bar and the maximum backpressure is &#60;400 bar.</li>
		<li>Confirm that the retention times are in a &#177;1 min window as follows: isoleucine 6.0 min, leucine 6.4 min, AMP 9.5 min, ADP 11.2 min, and ATP 12.7 min.</li>
		<li>Ensure the valley between leucine and isoleucine in the +132-&#62;86 transition is &#60; 20 % of the peak height.</li>
		<li>Ensure the difference in retention time AMP to ADP is 1.5 to 1.9 min, and the difference in retention time ADP to ATP is 1.1 to 1.9&#160;min.</li>
	</ol>
	</li>
	<li>Set up worklist
	<ol>
		<li>Start with a blank sample to minimize carry-over from the LC test mix.</li>
		<li>Run the samples in randomized order.</li>
		<li>End with a blank sample to clean the column for future experiments.</li>
	</ol>
	</li>
</ol>
6. Data pre-processing in automRm
NOTE: The following steps discuss conversion from .d to .mzML in msconvert, preparation of metabdb, loading of data and models and metabdb, and general strategies for manual peak review.
<ol>
	<li>Convert raw data from .d format to .mzML format using ProteoWizard20 with the following settings: binary encoding precision: 32-bit, MS Levels 1-2, Write Index selected, Use zlib compression selected.</li>
	<li>Start R.</li>
	<li>Install automRm21 according to the package documentation (only required once before first use).</li>
	<li>Start automRm GUI in R: automRm::automrm_gui()</li>
	<li>Process the batch of .d files in the tab Process &#62; Process Batch.
	<ol>
		<li>Select .xlsx file holding metabolite information. This file must match the LC-QQQ-MS method.</li>
		<li>Select .RData file holding peak picking ML model.</li>
		<li>Select .RData file holding the peak reporting ML model.</li>
		<li>Select the project folder containing .mzML files.</li>
		<li>Start data pre-processing by clicking Process Batch.</li>
	</ol>
	</li>
	<li>After the pre-processing is finished, open peakoverview.pdf to check the correct detection of chromatographic peaks. Optionally, load the automRmdata_Review.RData from the Review tab of the automRm GUI to manually modify peak filtering and peak integration.</li>
	<li>Open the peakinfo.xlsx from the project folder and check the data.
	<ol>
		<li>Check 13C internal standard signal in the tab 13CArea: Ensure that there are no systematic differences in the intensity values among experimental groups and between cell samples and debris samples. If such differences exist, only use data from the tabs NormArea or NormHeight for subsequent analysis; otherwise, use data from the tabs RawArea or RawHeight.</li>
		<li>Check the signal intensity of internal standards ATA and CLF in tab RawArea. Ensure that there are no systematic differences in the intensity of either compound among experimental groups.</li>
		<li>For each metabolite, compare the signal intensity of samples containing cells to the debris samples from the same cell suspension. Use an appropriate statistical test to identify metabolites in which samples containing cells have higher intensities than debris samples. Only include metabolites that pass this test in subsequent data analysis.</li>
	</ol>
	</li>
</ol>

Low-Input Metabolic Studies in Mice Cell Types

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferences+and+contaminants+encountered+in+modern+mass+spectrometry.">Interferences and contaminants encountered in modern mass spectrometry.</a> Analytica Chimica Acta. 627 (1), 71-81 (2008).</li><li>Kuonen, F., Touvrey, C., Laurent, J., Ruegg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fc+block+treatment,+dead+cells+exclusion,+and+cell+aggregates+discrimination+concur+to+prevent+phenotypical+artifacts+in+the+analysis+of+subpopulations+of+tumor-infiltrating+CD11b++myelomonocytic+cells.">Fc block treatment, dead cells exclusion, and cell aggregates discrimination concur to prevent phenotypical artifacts in the analysis of subpopulations of tumor-infiltrating CD11b+ myelomonocytic cells.</a> Cytometry. Part A The Journal of the International Society for Analytical Cytology. 77 (11), 1082-1090 (2010).</li><li>Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+specific+cell+population+by+fluorescence+activated+cell+sorting+(FACS).">Purification of specific cell population by fluorescence activated cell sorting (FACS).</a> Journal of Visualized Experiments. 41, 1546 (2010).</li><li>Broadhurst, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+and+considerations+for+the+use+of+system+suitability+and+quality+control+samples+in+mass+spectrometry+assays+applied+in+untargeted+clinical+metabolomic+studies.">Guidelines and considerations for the use of system suitability and quality control samples in mass spectrometry assays applied in untargeted clinical metabolomic studies.</a> Metabolomics. 14 (6), 72 (2018).</li></ol>

The authors declare no conflicts of interest.

The most critical steps for successful implementation of targeted metabolomics using this protocol are 1) a robust staining and gating strategy that will yield clean cell populations 2) precise handling of liquid volumes, 3) reproducible timing of all experimental steps, in particular all steps prior to metabolite extraction. Ideally, all samples belonging to one experiment should be processed and measured in one batch to minimize batch effects22. For larger experiments, we suggest collecting cell...

Metabolism is an essential biological process that occurs in all living cells. Metabolic processes involve a vast network of biochemical reactions that are tightly regulated and interconnected, allowing cells to produce energy and synthesize essential biomolecules1. To understand the function of metabolic networks, researchers measure the levels of small molecule intermediates within cells. These intermediates serve as important indicators of metabolic activity and can reveal critical insights into cellular function.
Mass spectrometry (MS) is the most popular choice for the specific detection of metabolites in complex samples1,2. Nuclear magnetic resonance (NMR) has advantages in the absolute quantification of compounds and structure elucidation, but MS can often resolve more components in complex mixtures such as biofluids or cell extracts. More often than not, MS is combined with prior separation of the compound by capillary electrophoresis (CE), gas chromatography (GC), or liquid chromatography (LC)3. The choice of separation platform is mostly driven by the range of target metabolites and the type of sample and, in a real-world setting, by the availability of machines and expertise. All three separation platforms have a broad and overlapping range of suitable metabolites but different limitations. Briefly, CE can only separate charged molecules and requires a lot of expertise to implement robust analysis of a large number of samples4. GC is limited to molecules that are small and apolar enough to evaporate before decomposing3. Considering all commercially available LC columns, any two metabolites can be separated by this technology5. However, many LC methods exhibit less resolving power than CE or GC methods of similar length.
The typical amount of starting material for metabolomics measurements is usually in the range of 5 x 105 to 5 x 107 cells per sample, 5-50 mg of wet tissue, or 5-50 &#181;L of body fluid6. However, it can be challenging to obtain such amounts of starting material when working with primary cells of rare cell types, such as for example hematopoietic stem cells (HSCs) or circulating tumor cells. These cells are often present in very low numbers and cannot be cultivated without compromising critical cellular features.
HSCs and multipotent progenitor cells (MPPs) are the least differentiated cells of the hematopoietic system and continuously produce new blood cells throughout an organism&#39;s life. The regulation of hematopoiesis is of clinical relevance in conditions such as leukemia and anemia. Despite their importance, HSCs and MPPs are among the rarest cells within the hematopoietic system. From a single mouse, typically, about 5000 HSCs can be isolated7,8,9. As traditional metabolomics methods require more input material, pooling cells from multiple mice was often necessary to analyze rare cell types10,11.
Here, we aimed to develop a protocol that enables the measurement of metabolites in as little as 5000 cells per sample to enable the generation of metabolomics data from the HSCs of a single mouse12. At the same time, this method allows to generate multiple replicates from a single mouse for more abundant cell types like lymphocytes. This approach reduces the number of animals required for a given project, thus contributing to the &#34;3R&#34; (reduction, replacement, refinement) of animal experiments.
Metabolites in cells can have very high turnover rates, often in the order of seconds13. However, preparing samples for fluorescence-activated cell sorting (FACS) can take hours, and FACS sorting itself can take minutes to hours, leading to potential alterations in the metabolome due to non-physiological conditions. Some of the reagents used in this protocol (such as ammonium-chloride-potassium [ACK] lysis buffer) can have similar effects. These conditions can cause cellular stress and impact the levels and ratios of metabolites within cells, leading to inaccurate or biased measurements of cellular metabolism14,15,16. The metabolic changes due to sample preparation are sometimes referred to as sorting artifacts. Long digestion protocols and harsh reagents that might be required to produce single-cell suspensions from hard or tough tissues can aggravate this issue. What changes can occur likely depends on the cell type and the processing condition. The precise nature of the changes remains unknown, as the metabolic state of the undisturbed cells in the living tissue cannot be measured.
The protocol presented here involves several key differences compared to traditional methods, namely the use of 5 g/L NaCl as a sheath fluid, sorting directly into extraction buffer, injecting large sample volumes on hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS), and utilizing targeted quantification, rigorous use of internal standards and background controls (Figure 1). This protocol has the potential to preserve differences among cell types and between drug treatment and vehicle control to a large extent12. Even for cultured cells, it compares favorably to alternative approaches, such as the more established centrifugation and manual removal of supernatant. However, as sorting artifacts may still occur, data must be interpreted with caution. Despite this limitation, the protocol represents a significant improvement in the field of metabolic profiling, allowing for more accurate and comprehensive measurements of cellular metabolism in rare primary cells12.
The ability to robustly measure broad metabolic profiles in rare primary cells opens the door to new experiments in biomedical research involving these cells. For example, metabolically mediated regulation in HSCs has been shown to impact dormancy self-renewal capacity, with implications for anemia and leukemia11,17. In patient-derived circulating tumor cells, differences in the expression of metabolic genes between tumor and adjacent cells have been shown18,19. This protocol now allows researchers to study these differences systematically on a metabolic level, which is generally regarded as closer to the cellular phenotype than gene expression.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>13C yeast extract</td>
			<td>Isotopic Solutions</td>
			<td>ISO-1</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile, LC-MS grade</td>
			<td>VWR</td>
			<td>83640.32</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK lysis buffer</td>
			<td>Gibco</td>
			<td>104921</td>
			<td>Alternatively: Lonza, Cat# BP10-548E</td>
		</tr>
		<tr>
			<td>Adenosine diphosphate (ADP)</td>
			<td>Sigma Aldrich</td>
			<td>A2754</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine monophosphate (AMP)</td>
			<td>Sigma Aldrich</td>
			<td>A1752</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine triphosphate (ATP)</td>
			<td>Sigma Aldrich</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Carbonate, HPLC grade</td>
			<td>Fisher Scientific</td>
			<td>A/3686/50</td>
			<td></td>
		</tr>
		<tr>
			<td>Atlantis Premier BEH Z-HILIC column (100 x 2.1 mm, 1.7 &#181;m)</td>
			<td>Waters</td>
			<td>186009982</td>
			<td></td>
		</tr>
		<tr>
			<td>B220-A647</td>
			<td>Invitrogen</td>
			<td>103226</td>
			<td></td>
		</tr>
		<tr>
			<td>B220-PE/Cy7</td>
			<td>BioLegend</td>
			<td>103222</td>
			<td>RRID:AB_313005</td>
		</tr>
		<tr>
			<td>CD11b-PE/Cy7</td>
			<td>BioLegend</td>
			<td>101216</td>
			<td>RRID:AB_312799</td>
		</tr>
		<tr>
			<td>CD150-BV605</td>
			<td>BioLegend</td>
			<td>115927</td>
			<td>RRID:AB_11204248</td>
		</tr>
		<tr>
			<td>CD3-PE</td>
			<td>Invitrogen</td>
			<td>12-0031-83</td>
			<td></td>
		</tr>
		<tr>
			<td>CD48-BV421</td>
			<td>BioLegend</td>
			<td>103428</td>
			<td>RRID:AB_2650894</td>
		</tr>
		<tr>
			<td>CD4-PE/Cy7</td>
			<td>BioLegend</td>
			<td>100422</td>
			<td>RRID:AB_2660860</td>
		</tr>
		<tr>
			<td>CD8a-PE/Cy7</td>
			<td>BioLegend</td>
			<td>100722</td>
			<td>RRID:AB_312761</td>
		</tr>
		<tr>
			<td>cKit-PE</td>
			<td>BioLegend</td>
			<td>105808</td>
			<td>RRID:AB_313217</td>
		</tr>
		<tr>
			<td>Dynabeads Untouched Mouse CD4 Cells Kit&#160;</td>
			<td>Invitrogen</td>
			<td>11415D</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSAria III</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gr1-PE/Cy7</td>
			<td>BioLegend</td>
			<td>108416</td>
			<td>RRID:AB_313381</td>
		</tr>
		<tr>
			<td>Heat sealing foil</td>
			<td>Neolab</td>
			<td>Jul-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoleucine</td>
			<td>Sigma Aldrich</td>
			<td>58880</td>
			<td></td>
		</tr>
		<tr>
			<td>JetStream ESI Source</td>
			<td>Agilent</td>
			<td>G1958B</td>
			<td></td>
		</tr>
		<tr>
			<td>Leucine</td>
			<td>Sigma Aldrich</td>
			<td>L8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Medronic acid</td>
			<td>Sigma Aldrich</td>
			<td>M9508-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol, LC-MS grade</td>
			<td>Carl Roth</td>
			<td>HN41.2</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fluka</td>
			<td>31434-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Sca1-APC/Cy7</td>
			<td>BioLegend</td>
			<td>108126</td>
			<td>RRID:AB_10645327</td>
		</tr>
		<tr>
			<td>TER119-PE/Cy7</td>
			<td>BioLegend</td>
			<td>116221</td>
			<td>RRID:AB_2137789</td>
		</tr>
		<tr>
			<td>Triple Quadrupole Mass Spectrometer</td>
			<td>Agilent</td>
			<td>6495B</td>
			<td></td>
		</tr>
		<tr>
			<td>Twin.tec PCR plate 96 well LoBind skirted</td>
			<td>Eppendorf</td>
			<td>30129512</td>
			<td></td>
		</tr>
		<tr>
			<td>UHPLC Autosampler</td>
			<td>Agilent</td>
			<td>G7157B</td>
			<td></td>
		</tr>
		<tr>
			<td>UHPLC Column Thermostat</td>
			<td>Agilent</td>
			<td>G7116B</td>
			<td></td>
		</tr>
		<tr>
			<td>UHPLC Pump</td>
			<td>Agilent</td>
			<td>G7120A</td>
			<td></td>
		</tr>
		<tr>
			<td>UHPLC Sample Thermostat</td>
			<td>Agilent</td>
			<td>G4761A</td>
			<td></td>
		</tr>
	</tbody>
</table>

targeted metabolomics on rare primary cells

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule intermediates. However, obtaining accurate and reliable measurements of cellular metabolism, particularly in rare cell types like hematopoietic stem cells, has traditionally required pooling cells from multiple animals. A protocol now enables researchers to measure metabolites in rare cell types using only one mouse per sample while generating multiple replicates for more abundant cell types. This reduces the number of animals that are required for a given project. The protocol presented here involves several key differences over traditional metabolomics protocols, such as using 5 g/L NaCl as a sheath fluid, sorting directly into acetonitrile, and utilizing targeted quantification with rigorous use of internal standards, allowing for more accurate and comprehensive measurements of cellular metabolism. Despite the time required for the isolation of single cells, fluorescent staining, and sorting, the protocol can preserve differences among cell types and drug treatments to a large extent.

Author Spotlight: Advancing Metabolomics Analysis of Rare Hematopoietic Stem Cells 

Targeted Metabolomics on Rare Primary Cells

The main goal of my lab is to maintain a healthy hematopoietic stem cell pull up in aging to avoid hematological malignancies, and we do so by performing dietary interventions. So in particular, we're interested to understand how dietary derived metabolites regulate stemness and also intracellular metabolites how they regulate the stem cell fate. Hematopoietic stem cells is a very rare population, meaning that we always face the challenge that we have not enough material to perform our experiments.But now with the establishment of single cell methods, and also with low input methods, we have achieved a new knowledge on how these stem cells are regulated. Hematopoietic stem cells is a very rare population and on top, they're very tiny. Now, with the methods that we have developed such, for example, low input metabolomics based on mass spec, we now can detect with very low number of cells, hundreds of metabolites, this now allowed us to understand what is the role of certain metabolites for stem cell regulation.In the past, metabolomics analysis of rare primary cells required many mice and days of work and sample preparation. With our new protocol, we need fewer mice and less time. Additionally, we have reduced the number of manual steps, so the whole protocol becomes more robust.For the future, we have planned to adapt our low input protocol to enable additional analysis and rare primary cells. For example, non-targeted metabolomics and lipidomics.

FACS sorting enables the isolation of clean populations of different cell types from the same cell suspension (Figure 2 and Figure 3). The specificity of this method relies on the staining of the different cell types with specific surface markers (for example, B cells and T cells from the spleen) or specific combinations of surface markers (for example HSCs and MPPs). Staining of intracellular markers typically requires permeabilization of the cell membrane. Thi...

Watch this Scientific Journal Video about Advancing Metabolomics Analysis of Rare Hematopoietic Stem Cells at JoVE.com

Here, we present a protocol to accurately and reliably measure metabolites in rare cell types. Technical improvements, including a modified sheath fluid for cell sorting and the generation of relevant blank samples, enable a comprehensive quantification of metabolites with an input of only 5000 cells per sample.

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule ...

targeted-metabolomics-on-rare-primary-cells

Research

JoVE Journal

Immunology and Infection

Mouse Splenic Single-Cell Suspension Generation and FACS Staining for Flow Cytometry-Based Sorting

FACS Sorting of Mouse Splenic Cells and Metabolite Analysis Using LC-MS

1.2K Views.  Max Planck Institute of Immunobiology and Epigenetics.  Here, we present a protocol to accurately and reliably measure metabolites in rare cell types. Technical improvements, including a modified sheath fluid for cell sorting and the generation of relevant blank samples, enable a comprehensive quantification of metabolites with an input of only 5000 cells per sample. 

Low-Input Metabolic Studies in Mice Cell Types

Summary