Fibrinogen anchors facilitates the micropatterning of hard-to-pattern cells. (A) Fibrinogen (doped with 10% fibrinogen-Alexa546), Con A (doped with 10% rhodamine–Con A), or fibrinogen–Con A (doped with 10% fibrinogen-Alexa546) were micropatterned at 50 µg/ml onto PLL-PEG–coated glass using deep-UV and a chromium mask. Coverslips were washed, and S2 cells were added for 1 h before addition of SiR-tubulin to label cells for 30 min. After washing, cells and micropatterns were imaged by spinning-disc confocal microscopy. While S2 cells do manage to adhere to larger Con A micropatterns, micropatterning efficiency of Con A is lower than that of fibrinogen (compare left panels), and therefore fibrinogen–Con A enables micropatterning of S2 cells onto small, single-cell micropatterns. Note that fluorescence in the “pattern” channel cannot be directly compared as Con A and fibrinogen are not functionalized with the same fluorophores. (B and C) Quantification of the effects seen in A (mean ± SEM; fibrinogen, n = 16; Con A, n = 14; fibrinogen–Con A, n = 11). Normalized micropatterned cell density (B) is significantly higher on small fibrinogen–Con A micropatterns compared with Con A or fibrinogen micropatterns, while the normalized nonspecific adhesion (C) is higher for fibrinogen and Con A than fibrinogen–Con A (see Materials and methods for details). Statistics in B were performed using a one-way ANOVA test followed by a Tukey post hoc test (P < 0.001), while statistics in C were performed using a Kruskal–Wallis test. n, number of fields of view analyzed. Scale bar, 100 µm. n.s., not significant.