Renner Patrick, Crone Michael, Kornas Matthew, Pioli KimAnh T, Pioli Peter D
Department of Investigative Medicine, Western Michigan University Homer Stryker M.D. School of Medicine, Kalamazoo 49008, USA.
Antib Ther. 2022 Jun 10;5(3):151-163. doi: 10.1093/abt/tbac013. eCollection 2022 Jul.
Antibody-secreting cells are terminally differentiated B cells that play a critical role in humoral immunity through immunoglobulin secretion along with possessing the potential to be long-lived. It is now appreciated that ASCs regulate multiple aspects of biology through the secretion of various cytokines. In this regard, ICFC is a key tool used to assess the presence of intracellular proteins such as cytokines and transcription factors.
Paraformaldehyde plus saponin or the eBioscience Foxp3/Transcription Factor Staining Buffer Set were used to evaluate the non-specific intracellular retention of phycoerythrin-containing antibody conjugates by ASCs.
We showed that the use of phycoerythrin-containing antibody conjugates led to a false interpretation of ASC intracellular protein expression compared with other cell types. This was mainly due to the inappropriate retention of these antibodies specifically within ASCs. Furthermore, we demonstrated how to reduce this retention which allowed for a more accurate comparison of intracellular protein expression between ASCs and other cell types such as B lymphocytes. Using this methodology, our data revealed that spleen ASCs expressed toll-like receptor 7 as well as the pro-form of the inflammatory cytokine interleukin-1β.
Increasing the number of centrifugation steps performed on ASCs post-fixation leads to inappropriate retention of phycoerythrin-containing antibody conjugates during ICFC.
抗体分泌细胞是终末分化的B细胞，通过分泌免疫球蛋白在体液免疫中发挥关键作用，并且具有长期存活的潜力。现在人们认识到，抗体分泌细胞通过分泌各种细胞因子来调节生物学的多个方面。在这方面，细胞内流式细胞术是评估细胞内蛋白质（如细胞因子和转录因子）存在的关键工具。
使用多聚甲醛加皂素或eBioscience Foxp3/转录因子染色缓冲液套装来评估抗体分泌细胞对含藻红蛋白抗体偶联物的非特异性细胞内滞留情况。
我们发现，与其他细胞类型相比，使用含藻红蛋白的抗体偶联物会导致对抗体分泌细胞内蛋白质表达的错误解读。这主要是由于这些抗体在抗体分泌细胞内的不适当滞留。此外，我们展示了如何减少这种滞留，从而能够更准确地比较抗体分泌细胞与其他细胞类型（如B淋巴细胞）之间的细胞内蛋白质表达。使用这种方法，我们的数据显示脾脏抗体分泌细胞表达Toll样受体7以及炎性细胞因子白细胞介素-1β的前体形式。
在固定后对抗体分泌细胞进行的离心步骤数量增加会导致在细胞内流式细胞术中含藻红蛋白的抗体偶联物不适当滞留。
