Las af application wizard v1

Manufactured by Leica

The LAS AF Application Wizard v1.7.0 is a software tool designed to assist users in the configuration and setup of Leica Microsystems' imaging and analysis systems. It provides a guided, step-by-step process to help users optimize their instrument settings for specific applications.

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LAS AF (255 citations) LAS AF v1.8 software (2 citations) Leica Application (2 citations)

6 protocols using las af application wizard v1

Detailed Live Cell FRET and FRAP Analysis

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LAS AF Application Wizard v1.7.0 (Leica) was used for detailed analyses of probes, including live cell acceptor photobleaching FRET (FRET-AB) experiments and fluorescence recovery after photobleaching (FRAP) experiments. We bleached the acceptor in the whole cell and calculated the efficiency of FRET. The recovery curve was used to estimate probe activities.

Zhang J., Wang Y., Zheng Z., Sun X., Chen T., Li C., Zhang X, & Guo J. (2019). Intracellular ion and protein nanoparticle-induced osmotic pressure modify astrocyte swelling and brain edema in response to glutamate stimuli. Redox Biology, 21, 101112.

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Quantifying Protein Interactions via FRET-AB and FRAP

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We applied LAS AF Application Wizard v1.7.0 (Leica) for detailed analyses of probes, including live cell acceptor photobleaching FRET (FRET-AB) experiments and fluorescence recovery after photobleaching (FRAP) experiments. The acceptor of the whole cell was bleached and then we calculated the efficiency of FRET. The constructed recovery curve was used to estimate probe activities.

Zheng Z., Wang T., Chen J., Qiu H., Zhang C., Liu W., Qin S., Tian J, & Guo J. (2021). Inflammasome-Induced Osmotic Pressure and the Mechanical Mechanisms Underlying Astrocytic Swelling and Membrane Blebbing in Pyroptosis. Frontiers in Immunology, 12, 688674.

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Evaluating Occludin and ZO1 Probe Efficiency

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LAS AF Application Wizard v1.7.0 (Leica) was used to detect the effectiveness of the occludin and ZO1 probes. Acceptor photobleaching FRET (FRET-AB) mode was used after HBMEC cells were transfected with the probes. Donor and receptor exciting light were adjusted to ensure fluorescence intensity of the donor and receptor when the exciting light was as low as possible. The whole cell was selected as the ROI, and the receptor exciting light intensity was adjusted to 100% to bleach the receptor. The ratio of the donor fluorescence intensity before and after bleaching was used to evaluate the FRET-AB efficiency of the probes.

Li C., Chen L., Wang Y., Wang T., Di D., Zhang H., Zhao H., Shen X, & Guo J. (2021). Protein Nanoparticle-Related Osmotic Pressure Modifies Nonselective Permeability of the Blood–Brain Barrier by Increasing Membrane Fluidity. International Journal of Nanomedicine, 16, 1663-1680.

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Fluorescence Recovery After Photobleaching

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Fluorescence recovery after photobleaching (FRAP) experiments was performed using LAS AF Application Wizard v1.7.0 (Leica). The ROI was selected on cells, 458 nm or 514 nm was chosen, and the exciting light intensity was adjusted to 100%. The ratio of fluorescence intensity of ROI before and 500 seconds after photobleaching was used to calculate the FRAP recovery rate of the probes.

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Fluorescence Recovery Kinetics Analysis

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Fluorescence recovery after photobleaching (FRAP) experiments were performed using LAS AF Application Wizard v1.7.0 (Leica). The ROI was selected on cells, 458 nm or 514 nm was chosen, and the exciting light intensity was adjusted to 100%. The ratio of uorescence intensity of ROI before and 500 seconds after photobleaching was used to calculate the FRAP recovery rate of the probes.

Li C., Chen L., Wang Y., Wang T., Di D., Zhang H., Zhao H., Shen X., & Guo J. (2020). Protein Nanoparticle Osmotic Pressure Modifies Nonselective Permeability of the Blood Brain Barrier by Increasing Membrane Fluidity.

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Measuring Occludin and ZO1 Probe Efficiency

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LAS AF Application Wizard v1.7.0 (Leica) was used to detect the effectiveness of the occludin and ZO1 probes. Acceptor photobleaching FRET (FRET-AB) mode was used after HBMEC cells were transfected with the probes. Donor and receptor exciting light was adjusted to ensure uorescence intensity of the donor and receptor when the exciting light was as low as possible. The whole cell was selected as the ROI, and the receptor exciting light intensity was adjusted to 100% to bleach the receptor. The ratio of the donor uorescence intensity before and after bleaching was used to evaluate the FRET-AB e ciency of the probes.

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