20October25_PSC051_PCLS

Plate Layout SYK PCLS Experiment 3 - October 20, 2025 Plate 1 - 0.1 R406µm Treated Plate (500ul of DMSO in 50ml media, 1%DMSO) ATP D1 ATP D3 ATP D5 Snap (Flex) D1 Snap (Flex) D3 Snap (Flex) D5 ATP D1 ATP D3 ATP D5 Snap (Flex) D1 Snap (Flex) D3 Snap (Flex) D5 FFPE D1 FFPE D3 FFPE D5 Snap (Flex) D1 Snap (Flex) D3 Snap (Flex) D5 FFPE D1 FFPE D3 FFPE D5 Untreated Control FFPE D1 Untreated Control FFPE D3 Untreated Control FFPE D5 Plate 2 - 0.01 R406µm Treated Plate (500ul of DMSO in 50ml media, 1%DMSO) ATP D1 ATP D3 ATP D5 Snap (Flex) D1 Snap (Flex) D3 Snap (Flex) D5 ATP D1 ATP D3 ATP D5 Snap (Flex) D1 Snap (Flex) D3 Snap (Flex) D5 FFPE D1 FFPE D3 FFPE D5 Snap (Flex) D1 Snap (Flex) D3 Snap (Flex) D5 FFPE D1 FFPE D3 FFPE D5 Untreated Control FFPE D1 Untreated Control FFPE D3 Untreated Control FFPE D5 ATP Assay Dead Cell Control Protocol - Based on concentrations suggested on Figure S1 of chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://pmc.ncbi.nlm.nih.gov/articl es/instance/2947864/bin/1011614107_pnas.1011614107_SI.pdf - Concentration: 0.3% Triton X-100 (3ul Triton X-1-00 + 997ul water) 1. Prepare 0.3% Triton X-100 in an eppendorf tube, ensure most Triton X-100 is dissolved, vortex if necessary. 2. Place PCLS slice in the eppendorf tube. 3. Vortex to mix. 4. Let sit at room temperature for 15 minutes, vortex periodically. 5. Pick up the slice using forceps and use ATP assay. Day 1 Raw Plate Readout - Temperature at time of reading: 22.6°C Standard Curve - Background: average of column 4 rows A and B [ATP] Luminescence 100 2853820 10 1491387 1 804063 0.1 408320 0.01 211695 0.001 109700.5 0.0001 57300.5 0.00001 29022.5 Results [ATP] Dead Cell 0, 0 Control 0.0041, 0.0011 SB 0.00093, 0.0107 0.1μM R406 0.0083, 0.0015 0.01μM R406 0.025, 0.070 Day 3 Raw Plate Readout - Temperature at time of reading: 22.2°C Standard Curve - Background: average of column 4 rows A and B [ATP] Luminescence 100 2300370.5 10 1266532.5 1 676588.5 0.1 364740 0.01 184356.5 0.001 100364 0.0001 55503 0.00001 25476.5 Results [ATP] Dead Cell 0, 0 Control 0.060, 0.038 SB 0.066, 0.060 0.1μM R406 0.0106, 0.032 0.01μM R406 0.0029, 0.00015 Day 5 Raw Plate Readout - Temperature at time of reading: 22.4°C Standard Curve - Background: average of column 4 rows A and B [ATP] Luminescence 100 2801814 10 1642160 1 859178.5 0.1 444179.5 0.01 236073 0.001 126349 0.0001 62321.5 0.00001 29447 Results [ATP] Dead Cell 0, 0 Control 0.015, 0.008 SB 0.19, 0.096 0.1μM R406 0.064, 0.057 0.01μM R406 0.002, 0.022