Figure S3.
Fibrinogen anchors improves selectivity and homogeneity of micropatterns on PEG-silane surfaces.(A) NeutrAvidin-DyLight-550 (50 µg/ml) was micropatterned on PLL-PEG–coated glass using LIMAP. Alternatively, fibrinogen-biotin was micropatterned with identical UV exposure, pattern shape, and protein concentration, followed by the addition of NeutrAvidin-Dylight-550 and imaging by TIRFM. (B) Quantification of amount of micropatterned protein (left), pattern selectivity (middle), and homogeneity (right) in the sample presented in A (mean ± SEM). Statistics were performed using a Mann–Whitney test for the left and right panels, and a Student’s t test for the middle panel. Fibrinogen-biotin significantly improves the micropatterning of NeutrAvidin-Dylight-550. (C) NeutrAvidin, NeutrAvidin-Dylight-550, or fibrinogen-biotin were micropatterned on PEG-silane–coated glass using LIMAP with identical UV exposure, pattern shape, and protein concentration (50 µg/ml). After pattern quenching and washing, BSA-biotin-Alexa647 (5 µg/ml) was added for 5 min. The sample was then washed, and BSA-biotin-Alexa647 fluorescence was imaged by TIRFM (NeutrAvidin, NeutrAvidin-Dylight-550, and fibrinogen-biotin samples). Alternatively, fibrinogen-biotin was micropatterned as above, and NeutrAvidin (or NeutrAvidin-Dylight-550) was added before addition of BSA-biotin-Alexa647 and TIRFM imaging. Two different exposures were used for each sample (top versus bottom line) so that each lane could be represented with the same dynamic range. (D) Quantification of the amount of micropatterned protein (left), micropattern selectivity (middle), and homogeneity (right) in the sample presented in C (mean ± SEM). Statistics were performed using a one-way ANOVA test followed by a Tukey post hoc test after log10 transformation of the data (P < 0.001). Fibrinogen-biotin enhances significantly the amount of BSA-biotin-Alexa647 patterned, as well as pattern selectivity and homogeneity. While fibrinogen-biotin efficiently improves the micropatterning of NeutrAvidin-Dylight-550 compared with direct micropatterning, this does not translate into improved micropatterning of BSA-biotin-Alexa647, likely because NeutrAvidin-Dylight-550 has lost some biotin-binding activity due to its fluorescent labeling. Scale bar, 10 µm. biot, biotin; n.s., not significant.

Fibrinogen anchors improves selectivity and homogeneity of micropatterns on PEG-silane surfaces. (A) NeutrAvidin-DyLight-550 (50 µg/ml) was micropatterned on PLL-PEG–coated glass using LIMAP. Alternatively, fibrinogen-biotin was micropatterned with identical UV exposure, pattern shape, and protein concentration, followed by the addition of NeutrAvidin-Dylight-550 and imaging by TIRFM. (B) Quantification of amount of micropatterned protein (left), pattern selectivity (middle), and homogeneity (right) in the sample presented in A (mean ± SEM). Statistics were performed using a Mann–Whitney test for the left and right panels, and a Student’s t test for the middle panel. Fibrinogen-biotin significantly improves the micropatterning of NeutrAvidin-Dylight-550. (C) NeutrAvidin, NeutrAvidin-Dylight-550, or fibrinogen-biotin were micropatterned on PEG-silane–coated glass using LIMAP with identical UV exposure, pattern shape, and protein concentration (50 µg/ml). After pattern quenching and washing, BSA-biotin-Alexa647 (5 µg/ml) was added for 5 min. The sample was then washed, and BSA-biotin-Alexa647 fluorescence was imaged by TIRFM (NeutrAvidin, NeutrAvidin-Dylight-550, and fibrinogen-biotin samples). Alternatively, fibrinogen-biotin was micropatterned as above, and NeutrAvidin (or NeutrAvidin-Dylight-550) was added before addition of BSA-biotin-Alexa647 and TIRFM imaging. Two different exposures were used for each sample (top versus bottom line) so that each lane could be represented with the same dynamic range. (D) Quantification of the amount of micropatterned protein (left), micropattern selectivity (middle), and homogeneity (right) in the sample presented in C (mean ± SEM). Statistics were performed using a one-way ANOVA test followed by a Tukey post hoc test after log10 transformation of the data (P < 0.001). Fibrinogen-biotin enhances significantly the amount of BSA-biotin-Alexa647 patterned, as well as pattern selectivity and homogeneity. While fibrinogen-biotin efficiently improves the micropatterning of NeutrAvidin-Dylight-550 compared with direct micropatterning, this does not translate into improved micropatterning of BSA-biotin-Alexa647, likely because NeutrAvidin-Dylight-550 has lost some biotin-binding activity due to its fluorescent labeling. Scale bar, 10 µm. biot, biotin; n.s., not significant.

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