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. Author manuscript; available in PMC: 2020 May 1.
Published in final edited form as: Biotechnol Bioeng. 2019 Feb 19;116(5):994–1005. doi: 10.1002/bit.26916

Figure 2 – Validation of Biopolymer Coatings.

Figure 2 –

Expression and cell-surface localization of biopolymer coatings was validated for the new, engineered 293-F cell lines. A, Representative confocal microscopy images of stable suspension adapted human embryonic kidney 293 (293-F) cell lines – wild type (w.t.), or stably expressing the Mucin-0, Mucin-135, or Mucin-270 biopolymer. Images show the cell membrane (shown in blue, CF633 Wheat Germ Agglutinin, WGA), O-glycans covalently attached to the Mucin-135 and Mucin-270 biopolymers (shown in red, CF568 Peanut Agglutinin, PNA), and green-fluorescent protein (shown in green, GFP) which is co-expressed on the plasmid with the Mucin-0, Mucin-135 and Mucin-270 biopolymer. B, Representative flow cytometry histograms showing the polydisperse population of biopolymer expressing cell lines compared to w.t. cells, y-axis is scaled to show the population distribution of GFP positive cells. >50,000 cells per histogram. C, Quantification of the percent of cells which are GFP positive for each cell line. Cells with GFP signal above the gray line in Fig. 2B were considered GFP positive. Mean and S.D. are shown, >50,000 cells per sample, n = 4. D, Representative immunoblot (left) and lectin blot (right) of whole cell lysates for each generated stable cell line compared to w.t. cells, n = 3. E, Viable cell concentration determined by hemocytometer counting with trypan blue exclusion, n = 3. F, GFP signal of Mucin-270 cells after induction of expression at t = 0 hr, measured by flow cytometry, n = 3, >15,000 cells per sample. G, Agarose gel showing polymerase chain reaction (PCR) product of Mucin-270 gene from DNA extracted from non-transfected cells (Mock), w.t. cells transiently transfected (Transient), or cells with the Mucin-270 gene incorporated in the genome and cultured for 2 months (2 mo.) or 12 days (12 d) after gentamycin selection. Star indicates the predicted molecular weight of Mucin-270 PCR product. #1 and #2 are biological replicates. Mean and S.D. shown, ns – not significant.