Name: Visualisatie van intracellulaire sialylering met klikchemie en expansiemicroscopie (Video) | JoVE Journal
Uploaded: 2025-02-07T13:00:00.000+00:00
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Description: Leer intracellulaire sialylering visualiseren met klikchemie en expansiemicroscopie. Deze methode combineert metabolische labeling met fysieke sample-vergroting voor hoge resolutie zonder dure super-resolutieapparatuur.

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<meta charset="utf8"></head><body>Expansiemicroscopie voor het visualiseren van intracellulaire gesialyleerde glycanen via MOE

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</ol>

<table><tbody><tr><td>(+) Sodium L-ascorbate</td><td>Sigma Aldrich</td><td>11140</td><td></td></tr><tr><td>12 well cell culture plate</td><td>Corning</td><td>3513</td><td></td></tr><tr><td>Acrylamide</td><td>Sigma Aldrich</td><td>A8887</td><td></td></tr><tr><td>Acrylic acid N-hydroxysuccinimide ester&nbsp;</td><td>Sigma Aldrich</td><td>A8060</td><td></td></tr><tr><td>Alexa Fluor 488 alkyne&nbsp;</td><td>Jena Bioscience</td><td>CLK-1277-5</td><td></td></tr><tr><td>Alexa Fluor 546 goat anti-mouse IgG</td><td>Invitrogen</td><td>A11003</td><td></td></tr><tr><td>Amonium persulfate</td><td>Sigma Aldrich</td><td>9913</td><td></td></tr><tr><td>Bis-Acrylamide</td><td>Sigma Aldrich</td><td>146072</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A7906</td><td></td></tr><tr><td>BTTAA</td><td>Jena Bioscience</td><td>CLK-067-100</td><td></td></tr><tr><td>Centrifugation tube 2 mL</td><td>EPPENDORF</td><td>30120094</td><td></td></tr><tr><td>Chloroquine diphosphate salt</td><td>Sigma Aldrich</td><td>C6628</td><td></td></tr><tr><td>Conical tube 15 mL</td><td>Falcon</td><td>352097</td><td></td></tr><tr><td>cover slips 12 mm #1</td><td>epredia</td><td>CB00120RA120MNZ0</td><td></td></tr><tr><td>cover slips 32 mm #1</td><td>epredia</td><td>CB00320RA140MNZ0</td><td></td></tr><tr><td>CuSO4</td><td>Sigma Aldrich</td><td>209198</td><td></td></tr><tr><td>DMEM high glucose medium&nbsp;</td><td>Dutscher</td><td>L0104-500</td><td></td></tr><tr><td>Dulbecco's Phosphate Buffered Saline (PBS)&nbsp;</td><td>Dutscher</td><td>L0615-500</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>biowest</td><td>S1810-500</td><td></td></tr><tr><td>Fibroblast 533T</td><td>-</td><td>-</td><td>Verzameld van een gezond individu</td></tr><tr><td>FIJI ImageJ 2.9.0</td><td>-</td><td>-</td><td></td></tr><tr><td>Gelatin</td><td>Bio-RAD</td><td>170-6537</td><td></td></tr><tr><td>Guanidine HCl</td><td>Sigma Aldrich</td><td>50950</td><td></td></tr><tr><td>HeLa cells</td><td>ATCC</td><td>CCL-2</td><td></td></tr><tr><td>Hoechst 33342</td><td>Sigma Aldrich</td><td>14533</td><td></td></tr><tr><td>Imaris 10.2</td><td>-</td><td>-</td><td></td></tr><tr><td>K2HPO4</td><td>Euromedex</td><td>PB0447-B</td><td>Watervrij</td></tr><tr><td>LSM 780 Confocal Microscopy&nbsp;</td><td>Zeiss</td><td>-</td><td></td></tr><tr><td>MCF7</td><td>ATCC</td><td>HTB-22</td><td></td></tr><tr><td>N-acetylmannosamine (ManNAc)</td><td>BIOSYNTH</td><td>MA05269</td><td></td></tr><tr><td>NaCl</td><td>Carlo Erba</td><td>479687</td><td></td></tr><tr><td>N-azidoacetylmannosamine (ManNAz)</td><td>BIOSYNTH</td><td>MA46002</td><td></td></tr><tr><td>Objectif "Plan-Apochromat" 63x/1,4 Oil DIC M27</td><td>Zeiss</td><td>420782-9900-799&nbsp;</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS) 10x</td><td>Euromedex</td><td>ET330</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma Aldrich</td><td>P2308</td><td>from&nbsp;Tritirachium album</td></tr><tr><td>purified mouse GM130 antibody</td><td>BD Bioscience</td><td>610822</td><td>50 &micro;g</td></tr><tr><td>Sodium acrylate&nbsp;</td><td>Sigma Aldrich</td><td>408220</td><td></td></tr><tr><td>T75 Flask</td><td>Corning</td><td>430641</td><td></td></tr><tr><td>TEMED</td><td>Sigma Aldrich</td><td>T9281</td><td></td></tr><tr><td>tris Acetate EDTA (TAE) 10x</td><td>Euromedex</td><td>EU0202-B</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X-100</td><td></td></tr><tr><td>Trypan Blue</td><td>Dutscher</td><td>702630</td><td></td></tr><tr><td>Trypsine-EDTA 1x</td><td>Dutscher</td><td>L0930-100</td><td></td></tr></tbody></table>

visualisatie van intracellulaire sialylering met klikchemie en expansiemicroscopie

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Hier wordt een nieuwe versie van expansiemicroscopie (ExM) gepresenteerd, Magnify, die is aangepast voor maximaal 11-voudige expansie, met behoud van een uitgebreide reeks biomolecuulklassen en compatibel is met een breed scala aan weefseltypen. Het maakt de ondervraging van de nanoschaalconfiguratie van biomoleculen mogelijk met behulp van conventionele diffractie-beperkte microscopen.

Universele moleculaire retentie met 11-voudige expansiemicroscopie

Er wordt een uniek, uitgebreid protocol gepresenteerd voor het genereren van gedesialyleerde humane monocyt-afgeleide dendritische cellen (mo-DC's) uit geïsoleerde perifere mononucleaire bloedcellen (PBMC's) met behulp van een sialidasebehandeling. Verder worden methoden beschreven om de fenotypische en functionele karakterisering van mo-DC's te beoordelen en te evalueren hoe behandeling met sialidase het rijpingsniveau van mo-DC's verbetert.

Generatie van monocyt-afgeleide dendritische cellen met verschillende gesialyleerde fenotypes

Leer intracellulaire sialylering visualiseren met klikchemie en expansiemicroscopie. Deze methode combineert metabolische labeling met fysieke sample-vergroting voor hoge resolutie zonder dure super-resolutieapparatuur.

Visualisatie van intracellulaire sialylering met klikchemie en expansiemicroscopie

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