Title: Supporting information for "Protein segregation and conformation in antigen-receptor triggering: a quantitative fluorescence microscopy study" Author: Jane Humphrey Email: janehumphrey@outlook.com Title of associated thesis: Protein segregation and conformation in antigen-receptor triggering: a quantitative fluorescence microscopy study DOI of associated thesis: 10.17863/CAM.70765 Supervisor: Prof. Sir David Klenerman Department: Chemistry University: Cambridge Date: 2014-2021 Licence: CC BY VIDEOS Video_01.avi Protein distribution in B cells. Three-channel video of A20 cells landing on an SLB coated with HEL-WT. HEL-WT is shown in orange, the BCR in red and CD45 in blue. Speed is 50x. Scale bar is 5 um. HEL-WT is labelled with carboxyrhodamine 110 and the BCR with tetramethylrhodamine; CD45 is labelled with a Fab, which is labelled at random lysine residues with Alexa Fluor 647. See section 3.2.1 of the associated thesis. Video_02.avi Protein distribution in B cells. Three-channel video of A20 cells landing on an SLB coated with HEL-XL. HEL-XL is shown in orange, the BCR in red and CD45 in blue. Speed is 50x. Scale bar is 5 um. HEL-XL is labelled with carboxyrhodamine 110 and the BCR with tetramethylrhodamine; CD45 is labelled with a Fab, which is labelled at random lysine residues with Alexa Fluor 647. See section 3.2.1 of the associated thesis. Video_03.avi Intracellular calcium release in the presence of DGS-NTA(Ni). Video of Jurkat cells attaching to a 5% DGS-NTA(Ni) SLB. Speed is 50x. Scale bar is 50 um. Cells are labelled with the calcium indicator Fluo-4. See section 4.2.3 of the associated thesis. Video_04.avi Intracellular calcium release in the absence of DGS-NTA(Ni). Video of Jurkat cells rolling on a 100% POPC SLB. Speed is 50x. Scale bar is 50 um. Cells are labelled with the calcium indicator Fluo-4. See section 4.2.3 of the associated thesis. Video_05.avi Intracellular calcium release in the presence of DGS-NTA(Ni). Tracking of Jurkat cells on a 5% DGS-NTA(Ni) SLB. The images have been filtered in preparation for analysis. Speed is 50x. Cells are labelled with the calcium indicator Fluo-4. See section 4.2.3 of the associated thesis. Video_06.avi Intracellular calcium release in the absence of DGS-NTA(Ni). Tracking of Jurkat cells on a 100% POPC SLB. The images have been filtered in preparation for analysis. Speed is 50x. Cells are labelled with the calcium indicator Fluo-4. See section 4.2.3 of the associated thesis. Video_07.avi T cells rolling in the absence of protein. Video of Jurkat cells rolling on a 0.5% DGS-NTA(Ni) SLB with 5 mM imidazole. CD45 is shown in blue. Speed is 10x. Scale bar is 5 um. CD45 is labelled with the Fab of GAP 8.3, which is labelled at random lysine residues with Alexa Fluor 488. See section 6.2.1 of the associated thesis. Video_08.avi Protein distribution in T cells. Two-channel video of Jurkat cells landing on an SLB coated with rCD2-S. rCD2-S is shown in orange and CD45 in blue. Speed is 50x. Scale bar is 5 um. rCD2-S is labelled at random lysine residues with Alexa Fluor 647; CD45 is labelled with the Fab of GAP 8.3, which is labelled at random lysine residues with Alexa Fluor 488. See section 6.2.1 of the associated thesis. Video_09.avi Protein distribution in T cells. Two-channel video of Jurkat cells landing on an SLB coated with rCD2-WT. rCD2-WT is shown in orange and CD45 in blue. Speed is 50x. Scale bar is 5 um. rCD2-WT is labelled at random lysine residues with Alexa Fluor 647; CD45 is labelled with the Fab of GAP 8.3, which is labelled at random lysine residues with Alexa Fluor 488. See section 6.2.1 of the associated thesis. Video_10.avi Protein distribution in T cells. Two-channel video of Jurkat cells landing on an SLB coated with rCD2-XL. rCD2-XL is shown in orange and CD45 in blue. Speed is 50x. Scale bar is 5 um. rCD2-XL is labelled at random lysine residues with Alexa Fluor 647; CD45 is labelled with the Fab of GAP 8.3, which is labelled at random lysine residues with Alexa Fluor 488. See section 6.2.1 of the associated thesis. Video_11.avi Single-particle tracking of Lck-XS. Video of Lck-XS diffusing in the plasma membrane of a Jurkat cell. Lck-XS incorporating Clover and mRuby2 was excited at 488 nm. Intensity in the donor (Clover) channel is shown. Speed is 1x. Scale bar is 5 um. See section 7.2.3 of the associated thesis. Video_12.avi Single-particle tracking of Lck-XS. Tracking of Lck-XS in the donor (Clover) channel. The images have been filtered in preparation for analysis. Speed is 1x. See section 7.2.3 of the associated thesis. SOFTWARE A PDF of each script or function is included along with the MATLAB file. To run the software, the following MATLAB Toolboxes are required: Symbolic Math; Signal Processing; Image Processing; Statistics and Machine Learning. The following functions are required: export_fig, written by Yair Altman (https://github.com/altmany/export_fig); pkfnd.m, cntrd.m and track.m, written by Daniel Blair and Eric Dufresne (http://site.physics.georgetown.edu/matlab). cellSegmentation.m This script automates cell selection, as described in section 2.7.1 of the associated thesis. proteinExclusion.m This script analyses the distribution of proteins at the interface between a cell and an SLB, as described in section 2.7.2 of the associated thesis. frap.m This script analyses FRAP data, as described in section 2.7.3 of the associated thesis. fcs.m This script analyses FCS data, as described in section 2.7.4 of the associated thesis. It is based on software written by Dr Paul D. Dunne. calciumRelease.m This script analyses cell movement and intracellular calcium release, as described in section 2.7.5 of the associated thesis. The initial tracking section is based on software written by Dr Aleks Ponjavic. acceptorPhotobleaching.m This script evaluates FRET using the acceptor photobleaching method, as described in section 2.7.6 of the associated thesis. particleTracking.m This script performs single-particle tracking, as described in section 2.7.8 of the associated thesis. It is based on software written by Dr Laura Weimann.