PhD Scientific Days 2024

Poster Session A - Molecular Medicine 1.

Isolation and Characterisation of Extracellular Vesicles from Lymph Nodes

Text of the abstract

Bernadett Réka Bodnár 1,2, Brachyahu Meir Kestecher *1,2,3, Sayam Ghosal 1,2, Edit Irén Buzás1,2,3, Xabier Osteikoetxea1,2
* Presenting author

Affiliation details:
1 HCEMM-SU Extracellular Vesicles Research Group, Budapest, Hungary Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary
2 Institute of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary
3 HUN-REN-SU Translational Extracellular Vesicle Research Group, Budapest

Introduction:
Extracellular vesicles (EVs) are lipid membrane-enclosed small particles that are released into the extracellular environment by all cells in nature. EVs can be obtained from cell conditioned media, biofluids and from the interstitial space of primary tissues. EV isolation from lymph nodes still remains a challenge, despite the well-described involvement of EVs in immune processes.

Aim:
Our aim here is to carry out the isolation and characterization of EVs from mouse lymph nodes.

Methods and Result:
C57BL/6 male mice were immunised with a stable emulsion of Complete Freund's Adjuvant alone or in combination with ovalbumin.
The inguinal and popliteal lymph nodes were isolated on day 9. EVs were separated from lymph nodes using differential centrifugation combined with size-exclusion chromatography and were further characterised using standard EV methodologies following the MISEV 2023 guidelines. Particle concentration was determined with Nanoparticle Tracking Analysis. Total protein concentration of the EV preparations was assessed with BCA assay and the total lipid content was measured with the SPV lipid assay. EV markers were analysed with a CytoFlex S Flow Cytometer. Within the large- and small-sized EV populations, we detected CD9, CD63 and Annexin V EV surface markers.

Conclusion:
Overall, here we present the isolation and characterisation EVs from the solid tissue of lymph nodes and the analysis of the effect immunisation on the isolated vesicles. Also, this work allows for further studies to understand immune processes and provides an opportunity to better understand the relationship between EVs and immune processes.

Funding:
This work was funded by the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 739593 and the Hungarian scientific research fund (OTKA) grant No FK147023