Programa de Doctorado en Ciencias Biomédicas orientación Inmunología, Centro Universitario de Ciencias de la Salud (CUCS), Universidad de Guadalajara, Guadalajara, México.
Instituto de Investigación en Enfermedades Crónico Degenerativas, Departamento de Biología Molecular y Genómica, Guadalajara, Centro Universitario de Ciencias de la Salud (CUCS), Universidad de Guadalajara, Guadalajara, Jalisco, México.
Technol Cancer Res Treat. 2023 Jan-Dec;22:15330338221150324. doi: 10.1177/15330338221150324.
Exosomes are the smallest of the extracellular vesicles and can contain a variety of different cargos, including nucleic acids, lipids, and proteins. Ultracentrifugation followed by electron microscopy has historically been used for the isolation and visualization of exosomes; Western blot and ELISA have also been used, but these techniques are only semiquantitative and are unable to distinguish different exosome markers in the same sample. To resolve some of these issues, we propose a modification of a bead-based flow cytometry method. Peripheral blood serum was mixed with a commercial exosome separation reagent and incubated for 30 min at 4°, centrifuged, exosome pellet was isolated and resuspended in PBS. Exosomes were then added to magnetic beads, incubated 18 h, then incubated with exosome-specific antibodies for 1 h. The resulting bead:exosome complexes were centrifuged and then washed, then washed again using a magnetic separator, resuspended in PBS, and analyzed via flow cytometry. Using commercial magnetic beads bound with anti-CD63, our protocol modifies starting conditions, washing steps, and magnetic separation and uses the FSC and SSC determination of the flow cytometer to result in increased yield and identification of the exosome populations of interest. Our modified protocol increased the yield of specific populations approximately 10-fold. The new protocol was used to identify exosomes positive for 2 immune checkpoint ligands in serum-derived exosomes from cervical cancer patients. We suspect that this protocol can also be used for the identification of other exosome proteins since we also quantified the exosome membrane-enriched tetraspanins CD9 and CD81. Identification of proteins rarely expressed in exosomes is complicated in this technique as serum is an inherently dirty source of exosomes, and great care must be taken in the washing and gating of the exosome:bead populations.
外泌体是最小的细胞外囊泡，可以包含多种不同的 cargo，包括核酸、脂质和蛋白质。超速离心结合电子显微镜一直用于外泌体的分离和可视化；Western blot 和 ELISA 也被用于检测，但这些技术只是半定量的，无法在同一样本中区分不同的外泌体标记物。为了解决其中的一些问题，我们提出了一种基于珠子的流式细胞术方法的改进。将外周血清与商业外泌体分离试剂混合，在 4°C 孵育 30 分钟，离心，分离出外泌体沉淀并悬浮在 PBS 中。然后将外泌体添加到磁性珠上，孵育 18 小时，然后用外泌体特异性抗体孵育 1 小时。将得到的珠：外泌体复合物离心并洗涤，然后再次使用磁分离器洗涤，悬浮在 PBS 中，并通过流式细胞术进行分析。使用与抗 CD63 结合的商业磁性珠，我们的方案修改了起始条件、洗涤步骤和磁分离，并使用流式细胞仪的 FSC 和 SSC 测定，导致感兴趣的外泌体群体的产量和鉴定增加。我们的改良方案使特定群体的产量增加了约 10 倍。该新方案用于鉴定宫颈癌患者血清衍生外泌体中 2 种免疫检查点配体阳性的外泌体。我们怀疑该方案也可用于鉴定其他外泌体蛋白，因为我们还定量了外泌体膜富集的四跨膜蛋白 CD9 和 CD81。由于血清是外泌体的固有污染源，因此在洗涤和外泌体：珠群体的门控中必须非常小心，因此在该技术中鉴定很少在外泌体中表达的蛋白质很复杂。
