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.

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

Author Spotlight: Advancing Metabolomics Analysis of Rare Hematopoietic Stem Cells 

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.

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 ...

targeted-metabolomics-on-rare-primary-cells

Research

JoVE Journal

Immunology and Infection

1.7K 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. 

Video: Author Spotlight: Advancing Metabolomics Analysis of Rare Hematopoietic Stem Cells 

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

&quot;Liver-on-a-Chip&quot; Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...