ROIs are distinguished based on whether they are (A,C,E) in cell-free regions of the sample (Cell-Free ROI) or (B,D,F) within cells (Cell ROI)

ROIs are distinguished based on whether they are (A,C,E) in cell-free regions of the sample (Cell-Free ROI) or (B,D,F) within cells (Cell ROI). black lines represent the average. Dashed lines represent a normalized fluorescence intensity of 1 1, i.e. the starting intensity prior to photobleaching. t = 0 s is usually defined as the start of fluorescence recovery.(PDF) pone.0158338.s003.pdf (166K) GUID:?2FD7884B-8D99-4F7D-A0F2-9E6A2F0C316C S4 Fig: (A,B) Normalized fluorescence intensities over time for photobleached ROIs of FITC-labeled (A) HSV8 IgG (HSV8 -gD”) and (B) HSV8 premixed with HSV-1 gD glycoprotein (HSV8 +gD) in pH-neutralized human CVM. ROIs were selected in cell-free regions of the samples. Thin grey lines represent individual measurements of unique ROIs, while solid black lines represent the average. Dashed lines represent a normalized fluorescence intensity of 1 1, i.e. the starting intensity prior to photobleaching. t = 0 s is usually defined as the start of fluorescence recovery. (C) Ratio of diffusivity in CVM (Dcvm) to diffusivity in PBS (Dpbs). (D) Unrecovered portion of fluorescence within the time level of measurement normalized to the initial bleached portion.(PDF) pone.0158338.s004.pdf (138K) GUID:?51CED1DD-D411-4C45-B75E-3F398C92D3EF S1 Text: Concentration of IgG vs. mucin molecules in human CVM. (PDF) pone.0158338.s005.pdf (88K) GUID:?03604242-CF73-4470-BE6B-8F1EF4241822 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Human cervicovaginal mucus (CVM) is usually a viscoelastic gel made up of a complex mixture of mucins, shed epithelial cells, microbes and macromolecules, such as antibodies, that together serve as the first line of defense against invading pathogens. Here, to investigate the affinity between IgG and different mucus AZ304 constituents, we used Fluorescence Recovery After Photobleaching (FRAP) to measure the diffusion of IgG in new, minimally modified CVM. We found that CVM exhibits substantial spatial variations that necessitate careful selection of the regions in which to perform FRAP. In portions of CVM devoid of cells, FRAP measurements using different IgG antibodies and labeling methods consistently demonstrate that both exogenous and endogenous IgG undergo quick diffusion, almost as fast as in saline, in good agreement with the quick diffusion of IgG in mid-cycle endocervical mucus that is largely devoid of cells. This quick diffusion indicates the interactions between secreted mucins and IgG must be very poor AZ304 and transient. IgG also accumulated in cellular debris and shed epithelial cells that experienced become permeable to IgG, which may allow shed epithelial cells to serve as reservoirs of secreted IgG. Interestingly, in contrast to cell-free regions of CVM, the diffusion of cell-associated IgG was markedly slowed, suggesting greater affinity between IgG and cellular constituents. Our findings Rabbit Polyclonal to GABBR2 contribute to an improved understanding of the role of IgG in mucosal protection against infectious diseases, and may also provide a framework for using FRAP to study molecular interactions in mucus and other complex biological environments. Introduction The human vaginal epithelium is coated with a layer of viscoelastic mucus gel comprised of long and greatly glycosylated mucin molecules that are cross-linked, entangled and bundled to form a porous network with large mesh spacings (common ~340 70 nm [1]), much larger than antibodies such as IgG and most mammalian viruses. Mucus secretions in the vagina are derived primarily from mucin-secreting glands in the endocervical canal [2]. As cervical mucus enters the vagina, it is altered by fluid and ion exchange [3,4], secreted or transudated antibodies, shed epithelial cells and vaginal microbiota [5,6]. As AZ304 a result, mucus overlaying the vaginal epithelium is frequently referred to as cervicovaginal mucus (CVM) to emphasize both its origin and the unique physicochemical properties that differentiate CVM from endo-cervical mucus. CVM not only serves as a lubricant minimizing physical trauma during coitus, but also provides an unstirred adherent layer of viscoelastic gel that can slow or prevent pathogens in semen from directly contacting the vaginal epithelium. Although native, acidic CVM was previously shown capable of trapping viruses [1,7], this trapping potency is generally AZ304 lost when the pH of CVM.