Name: Mitochondrial Preparation from Microglia for Glycan Analysis (Video) | JoVE Journal
Uploaded: 2025-05-30T14:10:00.000+00:00
Duration: 6 min 40 s
Description: Learn mitochondrial isolation from BV-2 microglial cells for glycan analysis using IR-MALDESI mass spectrometry. This protocol enables study of neuroimmune interactions in brain disorders.

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5 minutes

The authors would like to thank Seth Eisenberg, graduate student in Muddiman Lab at NCSU, for his help with video recording of mass spectrometric protocol. This research was supported in part by the School of Engineering Innovation Fellows Program at the University of Alabama at Birmingham, AG068309 to D.J.T. and R01GM087964-12&#160;to D.C.M. The schematics in this manuscript were drawn using BioRender.

1. BV2 Microglial cell line culture
<ol>
	<li>Maintain the BV-2 cells (microglial cells derived from C57BL/6 mice) in DMEM low glucose media supplemented with 10% Fetal Bovine serum (FBS), 1% Penicillium-Streptomycin (PenStrep), and 1% Non-essential Amino Acids (NEAA).</li>
	<li>Grow the cells in T-175 flasks and allow them to reach 70-80% confluency.</li>
	<li>Split the cells by incubating with the cell dissociation enzymes for 5 min at 37 &#176;C in 5% CO2, followed by inactivation of the enzymes with an equal volume of cell media, and centrifugation of cells for 5 min at 500 &#215; g at room temperature (RT). 
	NOTE: The cell dissociation enzymes used here are gentler on BV2 cells than trypsin and can be used to protect the cell surface antigen expression.</li>
	<li>Aspirate the media, resuspend the cell pellet in 1 mL of growth media, and count the cells using trypan blue (see the Table of Materials). 
	NOTE: We used an automated cell counter for this step to count cells.</li>
	<li>To carry-out the mitochondrial isolation, proceed if the cell pellet contains 2 &#215; 107 (20 million cells/pellet).</li>
</ol>
2. Isolation of mitochondria from microglial cells
NOTE: Work quickly, keeping everything on ice throughout the procedure. The mitochondrial isolation kit used for mitochondrial isolation has three components: Reagents A (cell lysis buffer), Reagent B (stabilizing buffer), and Reagent C (mitochondrial wash buffer). Add protease inhibitors to reagent A and reagent C immediately before use.
<ol>
	<li>Pellet 2 &#215; 107 cells by centrifuging harvested cells in a 2.0 mL microcentrifuge tube at 500 &#215; g for 5 min. Carefully aspirate and discard the supernatant.</li>
	<li>Add 800 &#181;L of mitochondrial isolation reagent A (cell lysis buffer), vortex at medium speed for 5 s, and incubate the tube on ice for exactly 2 min. 
	NOTE: Do not exceed the 2 min incubation.</li>
	<li>Add 10 &#181;L of mitochondrial isolation reagent B (stabilizing buffer), vortex at maximum speed for 5 s, and incubate the tube on ice for 5 min, vortexing at maximum speed every minute.</li>
	<li>Add 800 &#181;L of mitochondria isolation reagent C (mitochondrial wash buffer), invert the tube several times to mix, and centrifuge the tube at 700 &#215; g for 10 min at 4 &#176;C. 
	NOTE: Do not vortex.</li>
	<li>Transfer the supernatant to a new 2.0 mL tube and spin at 3,000 &#215; g for 15 min at 4 &#176;C.</li>
	<li>Transfer the supernatant (cytosolic portions) to a new tube. The pellet contains the isolated mitochondria.</li>
	<li>Add 500 &#181;L of Mitochondria isolation reagent C to the pellet, and centrifuge at 12,000 &#215; g for 5 min.</li>
	<li>Use the pellet for protein quantification and processing or store the pellet at -80 &#176;C until further use.</li>
</ol>
3. Protein estimation using microBCA assay
NOTE: Protein estimation for this protocol can be performed using different reagents and assays. Quantification of cytosolic or mitochondrial proteins can be performed by normalizing against the total protein concentration used in the assay.
<ol>
	<li>Prepare bovine serum albumin (BSA) standards between 0 &#181;g/mL and 200 &#181;g/mL (0 &#181;g/mL, 0.5 &#181;g/mL, 1 &#181;g/mL, 2.5 &#181;g/mL, 5 &#181;g/mL, 10 &#181;g/mL, 20 &#181;g/mL, 40 &#181;g/mL, 200 &#181;g/mL) and blanks with lysis buffer only.</li>
	<li>Add 150 &#181;L of each standard into the flat-bottomed 96-well plate containing the samples.</li>
	<li>Mix 25 parts of Micro BCA Reagent MA (bicinchoninic acid (BCA) solution) and 24 parts Reagent MB (copper sulfate solution) with 1 part of Reagent MC (stabilizing buffer) (25:24:1, Reagents MA:MB:MC) to create a working reagent. Add 150 &#181;L of the mixed BCA reagent to each sample and standard.</li>
	<li>Incubate at 37 &#176;C for 2 h.</li>
	<li>Use a plate reader to measure the absorbance at 562 nm and quantify the protein concentration with the standard curve. Subtract the blank standard absorbance from the absorbance measurement of all other individual standards and unknown sample replicates to obtain the sample protein concentrations.</li>
</ol>
4. Mitochondrial preparation quality control (western blot)
<ol>
	<li>Resuspend the mitochondrial pellet in the radioimmunoprecipitation assay buffer (RIPA) buffer to perform protein quantification. Determine the protein concentration for each sample using the micro BCA assay.</li>
	<li>Denature the samples in sample buffer at 94 oC for 5 min.</li>
	<li>Load equal amounts (20 &#181;g) of mitochondrial protein to precast gels (see Table of Materials), along with the molecular weight marker.</li>
	<li>Run the gel for 50 min at 100 V. 
	NOTE: The running time may vary, so make sure to stop the gel when the proteins have reached the end of the gel indicated by the dye in the sample buffer.</li>
	<li>Transfer the proteins from the gel to a polyvinylidene fluoride (PVDF) membrane using the dry-transfer protocol in a western blot transfer system for 7 min at 20 V.</li>
	<li>Take the membrane out and block using the blocking solution (Table 1) for 1 h at room temperature.</li>
	<li>Add appropriate dilutions of primary antibody to the membrane in blocking buffer overnight at 4 oC (COXIV= 1:3,000, GAPDH =1:3,000). 
	NOTE: The absence of nuclear, endoplasmic reticulum (ER) contamination in the mitochondrial preparation can be tested by using additional markers like lamin (nuclear marker) and ERp57 (ER marker) in western blots.</li>
	<li>Wash the membrane for 3 x 15 min with the wash buffer (Table 1).</li>
	<li>Incubate the membrane with the appropriate dilution of HRP-conjugated secondary antibody in blocking buffer at room temperature for 1 h</li>
	<li>Wash the membrane for 3 x 15 min with the wash buffer.</li>
	<li>For developing, use a chemiluminescent substrate kit.</li>
	<li>Scan the membrane using a gel and membrane imager.</li>
</ol>
5. Mitochondrial protein isolation for N -glycan extraction from microglia
<ol>
	<li>Resuspend the isolated mitochondria in 50 &#181;L of protein isolation buffer (Table 1) and leave on ice for 20 min.</li>
	<li>Aspirate and dispense three times and leave 20 min on ice (vortex before use). If the mitochondrial pellet is not fully solubilized, add another 50 &#181;L of the isolation buffer and pool into the same tube.</li>
	<li>Centrifuge at 13,000 &#215; g for 10 min.</li>
	<li>Recover the supernatant, freeze at -80 oC for a minimum of 1 h, and dry as much as possible in a vacuum concentrator.</li>
	<li>Resuspend before glycan isolation using the PNGase digest buffer (Table 1) for IR-MALDESI.</li>
</ol>
6. Mitochondrial N -glycan preparation for IR-MALDESI
<ol>
	<li>Load 25-250 &#181;g of protein (in 250 &#181;L maximum volume) of isolated mitochondrial proteins onto a 10 kDa molecular weight cut-off (MWCO) filter.</li>
	<li>Reduce the protein samples to expose the glycan moieties by adding 2 &#181;L of 1 M dithiothreitol (DTT) to each sample in the filter.</li>
	<li>Dilute the sample with 200 &#181;L of PNGase digest buffer and vortex lightly to avoid disturbing the filter.</li>
	<li>Denature the mitochondrial proteins by incubating the sample at 56 &#176;C for 30 min.</li>
	<li>Use 50 &#181;L of 1 M iodoacetamide to alkylate the mitochondria to give a final concentration of ~200 mM, and incubate at 37 &#176;C for 60 min.</li>
	<li>To further concentrate the denatured mitochondrial methods, centrifuge the samples at 14,000 &#215; g for 40 min. Discard the flowthrough.</li>
	<li>Wash the sample with 100 &#181;L of PNGase digest buffer.</li>
	<li>Concentrate the glycoprotein on the filter at 14,000 &#215; g for 20 min and discard the flowthrough. Repeat the wash and concentrate step 2x, for a total of 3x, yielding a concentrate in the filter dead volume (~5 &#181;L). Discard all flowthrough.</li>
	<li>Discard the collection vial once the washes are complete. Use a new collection vial for all future eluents and washes.</li>
	<li>To cleave the glycans from denatured mitochondrial glycopatterns, transfer to a fresh collection vial and add 2 &#181;L of glycerol-free PNGase (75,000 units/mL) to the filter. Add 98 &#181;L of PNGase digest buffer, bringing the total volume to 100 &#181;L and mix by gently pipetting up and down on the filter.</li>
	<li>Incubate samples at 37 &#176;C for 18 h to enzymatically cleave all N-glycans from mitochondrial proteins.</li>
	<li>Elute the released mitochondrial N-glycans by centrifuging the sample at 14,000 &#215; g for 20 min at 20 &#176;C.</li>
	<li>Wash the mitochondrial glycans by adding 100 &#181;L of PNGase digest buffer to the filter and centrifuge at 14,000 &#215; g for 20 min at 20 &#176;C. Collect the wash containing any remaining N-glycans in the same collection vial as the eluent. Repeat 2x and remove the filter from the collection vial.</li>
	<li>Incubate the mitochondrial glycan samples in the -80 &#176;C freezer until frozen (30-60 min) and dry to completion at room temperature in a vacuum concentrator (4-6 h for 400 &#181;L). 
	NOTE: N-glycans may be stored at -20 &#176;C for up to 6 months prior to analysis.</li>
	<li>Resuspend the dried N-linked glycans in 50 &#181;L of LC/MS grade water directly before IR-MALDESI analysis.</li>
</ol>
7. Detection of released glycans by IR-MALDESI mass spectrometry
<ol>
	<li>Perform mass calibration each day of the experiment. Load the calibration solution onto a syringe pump and push through at a rate of 1.2 &#181;L/min. Apply a voltage of 3.5 kV to achieve a stable electrospray plume for mass calibration in both positive and negative modes.</li>
	<li>Pipette 5 &#181;L of resuspended mitochondrial glycans onto a sample spot on a Teflon microwell slide.</li>
	<li>Use a mid-IR laser operating at a wavelength of 2.97 &#181;m for ablation with an energy of 1.8 mJ per burst.</li>
	<li>Ionize and detect&#160;N-glycans in negative ionization mode. Use electrospray solvent consisting of 60% acetonitrile and 1 mM acetic acid to create a stable electrospray plume with a flow rate of 2 &#181;L/min at a voltage of 3.2 kV.</li>
	<li>To perform the analysis, couple IR-MALDESI to a HRAM mass spectrometer set to a mass resolving power of 240,000FWHM at m/z 200, analyzing between 500 and 2,000 m/z in negative ionization mode.</li>
	<li>Turn off automatic gain control (AGC) and set a fixed injection time of 90 ms. Use the EasyIC source for real-time internal calibration of every spectrum to achieve high mass measurement accuracy (MMA).</li>
</ol>
8. Mitochondrial N- glycan data analysis
<ol>
	<li>Manually identify the N-linked glycans by searching for monoisotopic masses and confirming isotopic distributions using the m/z spacing to determine doubly and triply charged ions that have a minimum ion flux threshold of 1,000 ions/s.</li>
	<li>Convert the raw mass spectra from m/z ratios to neutral monoisotopic masses.</li>
	<li>Upload the monoisotopic masses to an online oligosaccharide structure prediction tool to determine potential glycan compositions. Confirm annotations using an experimentally curated glycomic database20; ensure that each identification is within the margin of 2.5 ppm MMA, contains the core N-linked glycan structure (Hex3HexNAc2), and excludes pentose, KDN, or HexA monosaccharides.</li>
	<li>Draw the confirmed glycan structures using SNFG nomenclature21.</li>
	<li>Obtain the relative abundance of glycans from the raw mass spectra and normalize against the amount of mitochondrial protein in &#181;g to obtain ions/s/&#181;g.</li>
	<li>Use Chi-squared analysis to test the goodness of fit between the theoretical and experimental distributions of the N-linked glycans to determine the number of chlorine adducts. This allows direct determination of the number of sialic acids19.</li>
</ol>

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</ol>

The authors have no conflicts of interest to declare.

Microglia are the resident immune cells of the brain, and glycan modifications modulate the immunophenotype and function of microglia. These immune functions demand substantial cellular energy, which is predominantly supplied by mitochondria. Notably, the mitochondrial proteins also present glycan modifications, which have remained significantly understudied due to the technological challenges in investigating sub-cellular glycosylation. Most studies investigating mitochondrial glycosylation rely on lectin-based identifi...

Microglia are the dominant resident innate immune cells in the brain and account for 10-15% of cells in the adult brain1,2. They use their receptor repertoire to dynamically monitor the brain microenvironment and regulate the normal brain function to maintain brain homeostasis3. Microglia are very sensitive to the changes in their microenvironment and undergo changes in cell morphology, immunophenotype, and function with pathological conditions or various stimulations. Microglial activation states are influenced by the cellular energy demands required for their function, such as phagocytosis, cytokine production, or tissue repair. Therefore, cellular energy metabolism plays a crucial role in regulating changes in microglial function4. Microglial dysregulation leads to excessive release of pro-inflammatory cytokines (e.g., IL-1&#946;, TNF-&#945;) and reactive oxygen species (ROS), predisposing the brain to neuroinflammation5,6. Chronic microglial dysregulation and the resulting neuroinflammatory environment lays a foundation for neurodegeneration7.
The brain accounts for only 2% of body weight but 20% of the body&#39;s total energy consumption. Mitochondria are the primary source of energy in brain cells and act as key players in the pathogenesis of both acute and chronic brain disorders8. Previous studies have established a strong correlation between microglial activation and metabolic dysfunction in aging9 and age-related disorders such as Alzheimer&#39;s disease10,11, highlighting the pivotal role of mitochondria in cellular senescence and neurodegeneration. Impaired mitochondrial function leads to diminished energy production, elevated oxidative stress, and increased neuroinflammation during aging and age-related diseases.
While extensive research has elucidated the role of mitochondria in energy metabolism, aging, and brain disorders, the role of common post-translational modifications, such as glycosylation, in mitochondrial biology and function remains insufficiently explored. Glycosylation, the enzymatic addition of sugar moieties called glycans to proteins by glycosylation enzymes, is the most common post-translational modification in most brain cells, including microglia. Activated microglia modulate their immune function under inflammatory stimuli by regulating the intracellular or cell surface glycan expression12. The pro- and anti-inflammatory responses exhibited by microglia post-stimulation are also regulated by the glycans13. Mitochondrial proteins also have these glycan modifications, which regulate their function and localization. However, detailed analysis of the cell-specific mitochondrial glycosylation patterns in the microglia is lacking due to the technical challenges in investigating sub-cellular glycosylation. Despite the well-characterized roles of glycosylation in modulating the microglial phenotype, the role of glycans in modulating mitochondrial function and subsequently, cellular immunophenotype in microglia remains poorly understood.
Limited studies investigating mitochondrial protein glycosylation have focused primarily on lectin-based identification of glycosylation patterns. Lectins are glycan-binding proteins that bind biomolecular glycan moieties14,15, which lack the specificity and ability to provide detailed information about the glycan composition. Mass spectrometric modalities offer a detailed identification of the glycan compositions to overcome the analytical challenges presented by lectin analysis. One such modality, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI), employs a hybrid ionization strategy, using a mid-IR laser to resonantly excite water found in biological specimens16 to desorb the neutral species and subject them to an orthogonal electrospray plume, followed by analysis using a high-resolution accurate mass Orbitrap mass spectrometer. IR-MALDESI has been previously demonstrated for the direct analysis of tissue metabolites17, with distinct advantages of rapid analysis18, soft ionization method, and the predictability of sialic acid content of N-linked glycans based on the isotopic distribution patterns of chlorinated glycan adducts19. However, the adaptation of this platform for the direct analysis of sub-cellular glycans has not been demonstrated.
Here, we report a high-throughput protocol for mitochondrial isolation from microglial cells, isolation of mitochondrial N-glycans, and mitochondrial N-glycan detection and analysis using IR-MALDESI mass spectrometry. This protocol will be foundational in uncovering novel insights into the role of glycosylation in mitochondrial function, potentially identifying new therapeutic targets for neuroinflammatory and neurodegenerative disorders.

<table><tbody><tr><td>&lt;strong&gt;&lt;em&gt;Equipment&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amersham 600 imager</td><td>Cytvia&amp;nbsp;</td><td>29194217</td><td>Gel and membrane imager</td></tr><tr><td>Countess 3 automated cell counter</td><td>Fisher Scientific</td><td>X003SZ1LY9</td><td></td></tr><tr><td>Dry Bath Stdrd 4 blck 100-120V</td><td>Thermofisher scientific&amp;nbsp;</td><td>88870003</td><td></td></tr><tr><td>i-Blot2 Gel Transfer Device</td><td>Invitrogen</td><td>IB21001</td><td>Western blot transfer system</td></tr><tr><td>Inverted microscope</td><td>Cell Treat</td><td>04355223EA</td><td></td></tr><tr><td>Microplate reader</td><td></td><td>82050-760</td><td></td></tr><tr><td>Mini gel tank</td><td>Invitrogen</td><td>A25977</td><td></td></tr><tr><td>Open Air Rocker</td><td>Fisher Brand</td><td>88861025</td><td></td></tr><tr><td>Pipet boy&amp;nbsp;</td><td>BioTek</td><td>229310</td><td></td></tr><tr><td>Vortex mixer</td><td>Integra- VWR</td><td></td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Mitochondria isolation reagents&amp;nbsp;&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mitochondrial Isolation kit</td><td>Thermofisher scientific&amp;nbsp;</td><td>89874</td><td></td></tr><tr><td>Phosphotase Inhibitor</td><td>Thermofisher scientific&amp;nbsp;</td><td>1861274</td><td></td></tr><tr><td>Protease Inhibitor</td><td>Thermofisher scientific&amp;nbsp;</td><td>1861281</td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;N-glycan isolation and IR-MALDESI reagents&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetic acid&amp;nbsp;</td><td>Fisher Scientific</td><td>A11350</td><td>50% in ESI solvent</td></tr><tr><td>Acetonitrile&amp;nbsp;</td><td>Sigma Aldrich</td><td>34851-4L</td><td>1 mM in ESI solvent</td></tr><tr><td>Ammonium bicarbonate&amp;nbsp;</td><td>Fisher Scientific</td><td>A643500</td><td>100 mM</td></tr><tr><td>Calibration Solution</td><td>Thermofisher Scientific</td><td>A39239</td><td>Pierce FlexMix</td></tr><tr><td>Dithiothreitol&amp;nbsp;</td><td>Sigma Aldrich</td><td>AC426380100</td><td>1 M</td></tr><tr><td>Iodoacetamide</td><td>Sigma Aldrich</td><td>A322-10VL</td><td></td></tr><tr><td>LC/MS grade water</td><td>Thermofisher Scientific</td><td>047146.M6</td><td></td></tr><tr><td>PNGase F</td><td>Bulldog Bio</td><td>NZPP010</td><td>75000 U/mL, enzyme for N-glycan release&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;N-glycan isolation and IR-MALDESI consumables&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amicon centrifugal filters</td><td>Fisher Scientific&amp;nbsp;</td><td>UFC501024</td><td>10 kDa MWCO</td></tr><tr><td>Mass spectrometer</td><td>Orbitrap Exploris 240</td><td></td><td></td></tr><tr><td>Mid-IR Laser</td><td>JGM Associates, Burlington, MA, USA</td><td></td><td></td></tr><tr><td>Teflon microwell slide</td><td>Prosolia, Indianapolis, IN, USA</td><td></td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;N-glycan analysis softwares&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GlycoMod&amp;nbsp;</td><td>Expasy&amp;nbsp;</td><td></td><td>https://web.expasy.org/glycomod/</td></tr><tr><td>GlyConnect</td><td>Expasy&amp;nbsp;</td><td></td><td>https://glyconnect.expasy.org/</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Protein isolation and western blot consumables&amp;nbsp;&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Basix gel loading tips ( 10 &amp;micro;L)&amp;nbsp;</td><td>Basix</td><td>13-611-102</td><td></td></tr><tr><td>Basix gel loading tips ( 200 &amp;micro;L)&amp;nbsp;</td><td>Basix</td><td>13-611-116</td><td></td></tr><tr><td>Cell scrapper</td><td>VWR labs</td><td>14-388-100</td><td></td></tr><tr><td>i-Blot NC regular stacks</td><td>Invitrogen</td><td>IB23001</td><td></td></tr><tr><td>i-Blot2 PVDF Regular Stacks</td><td>Invitrogen</td><td>IB24001</td><td></td></tr><tr><td>10&amp;nbsp; &amp;micro;L micropipette</td><td>Fisher Scientific</td><td>FBE00010</td><td></td></tr><tr><td>20 &amp;micro;L micropipette</td><td>Invitrogen</td><td>FBE00020</td><td></td></tr><tr><td>200&amp;nbsp; &amp;micro;L micropipette&amp;nbsp;</td><td>Fisher Brand</td><td>FBE00200</td><td></td></tr><tr><td>1000 &amp;micro;L micropipette</td><td>Fisher brand</td><td>FBE01000</td><td></td></tr><tr><td>10&amp;nbsp; &amp;micro;L pipet tips&amp;nbsp;</td><td>VWR labs</td><td>76322-528</td><td></td></tr><tr><td>20&amp;nbsp; &amp;micro;L pipet tips</td><td>VWR labs</td><td>76322-134</td><td></td></tr><tr><td>200&amp;nbsp; &amp;micro;L pipet tips&amp;nbsp;</td><td>VWR labs</td><td>76322-150</td><td></td></tr><tr><td>1000&amp;nbsp; &amp;micro;L pipet tips</td><td>VWR labs</td><td>76322-154</td><td></td></tr><tr><td>Well plate&amp;nbsp;</td><td>Fisher brand</td><td>14-388-100</td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Protein isolation and western blot reagents&amp;nbsp;&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Actin antibody ( Host : Rabbit )</td><td>Cell Signaling Technologies</td><td>8457T</td><td></td></tr><tr><td>Anti-Rabbit IgG HRP Linked&amp;nbsp;</td><td>Cell Signaling Technologies</td><td>7074S</td><td></td></tr><tr><td>Bolt 4-12% Bis-Tris Plus</td><td>Invitrogen</td><td>NW04120BOX</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Fisher bioreagents</td><td>BP9700-100</td><td></td></tr><tr><td>COXIV antibody ( Host : Rabbit)</td><td>Cell Signaling Technologies</td><td>4844S</td><td></td></tr><tr><td>GAPDH antibody ( Host : Rabbit)</td><td>Cell Signaling Technologies</td><td>2118S</td><td></td></tr><tr><td>MicroBCA protein assay Kit</td><td>Thermofisher scientific&amp;nbsp;</td><td>23235</td><td></td></tr><tr><td>Nupage MOPS SDS Runing Buffer [20x]</td><td>Thermofisher scientific&amp;nbsp;</td><td>NP0001</td><td></td></tr><tr><td>PAGE Ruler prestained protein ladder</td><td>Thermofisher scientific&amp;nbsp;</td><td>815-968-0747</td><td>Dilution= Use 7&amp;nbsp; &amp;micro;L to load onto first well</td></tr><tr><td>Phosphate buffered saline&amp;nbsp;</td><td>Aniara Diagnostics</td><td>A12-9423-5</td><td>Prepare 1x PBS from 10x powder&amp;nbsp;</td></tr><tr><td>Pierce ECL Western Blotting Substrate</td><td>Thermofisher scientific&amp;nbsp;</td><td>32106</td><td>Chemiluminescent substrate kit</td></tr><tr><td>RIPA Buffer</td><td>Thermofisher scientific&amp;nbsp;</td><td>89901</td><td></td></tr><tr><td>Sample Buffer</td><td>Novex</td><td>B0007</td><td>The bolt LDS sample buffer is prepared in 3:1 ratio of sample to sample buffer</td></tr><tr><td>Tween-20</td><td>MP Biomedicals</td><td>TWEEN201</td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Tissue culture consumables&amp;nbsp;&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Countess Slides</td><td>Avantor</td><td>229411</td><td></td></tr><tr><td>Eppendorf tubes</td><td>Cell Treat</td><td>414004-265612-5884</td><td></td></tr><tr><td>2 mL aspirating pipet</td><td>Vista lab</td><td>5090-0010E</td><td></td></tr><tr><td>5 mL serological pipet&amp;nbsp;</td><td>Fisher Scientific</td><td>13-678-11D</td><td></td></tr><tr><td>10 mL serological pipet&amp;nbsp;</td><td>Basix</td><td>13-678-11E</td><td></td></tr><tr><td>25 mL serological pipet</td><td>Vista lab</td><td>FB012937</td><td></td></tr><tr><td>50 mL serological pipet</td><td>Vista lab</td><td>14955233</td><td></td></tr><tr><td>15 mL Conical tube</td><td>Avantor</td><td>229225A</td><td></td></tr><tr><td>50 mL conical tube</td><td>Cell treat</td><td>4190-0050</td><td></td></tr><tr><td>T-75 cm&lt;sup&gt;2&lt;/sup&gt; Tissue culture flask</td><td>Fisher Scientific</td><td>FB012937</td><td></td></tr><tr><td>T-180 cm&lt;sup&gt;2&lt;/sup&gt; Tissue culture flask</td><td>Fisher Scientific</td><td>FB012939</td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Tissue culture reagents&amp;nbsp;&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BV2 microglial cell line&amp;nbsp;</td><td>Creative Bioarray&amp;nbsp;</td><td>CSC-I2227Z</td><td>Immortalized Mouse Microglia (BV2) derived from C57/BL6 neonatal microglia&amp;nbsp;</td></tr><tr><td>Cell dissociation enzymes&amp;nbsp;</td><td>Thermofisher scientific&amp;nbsp;</td><td>12563029</td><td>TrypLE&amp;nbsp;</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM) Low Glucose Media&amp;nbsp;</td><td>Gibco</td><td>10567014</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Cytiva</td><td>SH30071.03HI</td><td></td></tr><tr><td>Minimum Essential Medium (MEM) Non-essential Amino Acids</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>Penicillium Streptomycin</td><td>Cytivia</td><td>SV30010</td><td></td></tr><tr><td>Phosphate buffer saline</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Trypan Blue stain 0.4%</td><td>Invitrogen</td><td>T10282</td><td></td></tr></tbody></table>

mitochondrial preparation from microglia for glycan analysis

Mitochondrial Preparation from Microglia for Glycan Analysis

Understanding the glycosylation patterns of mitochondrial proteins in microglia is critical for determining their role in neurodegenerative diseases. Here, we present a novel and high-throughput methodology for glycomic analysis of mitochondrial proteins isolated from cultured microglia. This method involves the isolation of mitochondria from microglial cultures, quality assessment of mitochondrial samples, followed by an optimized protein extraction to maximize glycan detection, and infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) high-resolution accurate mass (HRAM) mass spectrometry to provide detailed profiles of mitochondrial glycosylation.
This protocol emphasizes the importance of maintaining mitochondrial integrity during isolation and employs stringent quality control to ensure reproducibility, including measuring mitochondrial purity after extraction. This approach allows for the comprehensive profiling of glycosylation changes in microglial mitochondria under various experimental conditions in vitro, which offers insight into mitochondrial changes associated with neurodegenerative diseases. This approach could be adapted to other in vitro treatments, other cultured cell types, or primary cells. Through this standardized approach, we aim to advance the understanding of microglial mitochondrial glycans, contributing to the broader field of neurodegenerative research.

Figure 1 represents a schematic outline of the steps involved in the isolation of mitochondria from the BV2 microglial cell line for mass spectrometric glycan analysis. The reproducibility of mitochondrial protein isolation between different mitochondrial preparations from the same starting density of the microglial cells is represented in Figure 2, which shows no significant difference between the mitochondrial protein concentration estimated using the micro BC...

A protocol was developed for the preparation of purified mitochondria from microglial cells, isolation of mitochondrial proteins for N-glycan release, and rapid detection of subcellular, mitochondrial glycans using infrared matrix-assisted laser desorption electrospray ionization coupled to high-resolution accurate mass analyzer mass spectrometry.

Understanding the glycosylation patterns of mitochondrial proteins in microglia is critical for determining their role in neurodegenerative diseases. ...

mitochondrial-preparation-from-microglia-for-glycan-analysis

Research

JoVE Journal

Bioengineering

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions.  This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

The video demonstrates a technique for isolating microglia from the cortex of an adult mouse brain. First, the cortex undergoes segmentation and homogenization to remove neurons and other glial cells, preserving microglia. Following that, the cell suspension is exposed to anti-myelin magnetic microbeads and a magnetic field, effectively separating microglia and myeloid cells from myelin debris and cells containing myelin.

Isolation of Microglia from the Cortex of an Adult Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord.  The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Culturing Microglia from the Neonatal and Adult Central Nervous System

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Here, a protocol (MitoCeption) is presented to transfer mitochondria, isolated from human mesenchymal stem cells (MSC), to glioblastoma stem cells (GSC), with the goal of studying their biological effects on GSC metabolism and functions. A similar protocol can be adapted to transfer mitochondria between other cell types.

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

This study presents a method to analyze the morphology of mitochondria based on immunostaining and image analysis in mouse brain tissue in situ. It also describes how this allows one to detect changes in mitochondrial morphology induced by protein aggregation in Parkinson's disease models.

Histological Examination of Mitochondrial Morphology in a Parkinson's Disease Model

This protocol describes a cost-effective way to perform a glycolysis stress test to quantify metabolic reprogramming in primary mouse microglia. After isolation and inflammatory stimulation, extracellular acidification rate (ECAR) is measured using freshly prepared, pH-adjusted reagents, followed by protein normalization to enable accurate and reproducible assessment of glycolytic shifts.

Investigating Glycolysis in Primary Microglia Using Extracellular Flux Assay

Learn mitochondrial isolation from BV-2 microglial cells for glycan analysis using IR-MALDESI mass spectrometry. This protocol enables study of neuroimmune interactions in brain disorders.

Mitochondrial Preparation from Microglia for Glycan Analysis

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