Nexcelom 3D Plates for 3D Tumor Spheroid Analysis

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1 Nexcelom3D Ultra- low Attachment Treated Round Bottom Multi- well Plates for Single Spheroid Drug Screening Assays Products Nexcelom 3D 96- well Ultra- low attachment treated round bottom multi- well plates Clear round bottom, polystyrene, 96- well, individually packed with lid, sterile, for 3D spheroid formation and measurement Nexcelom 3D 384- well Ultra- low attachment treated round bottom multi- well plates Clear round bottom, polystyrene, 384- well, individually packed with lid, sterile, for 3D spheroid formation and measurement Cat# Plates/Case Price ULA- 96U $ ULA- 96U $ ULA- 96U $1, ULA- 384U $ ULA- 384U $ ULA- 384U $2, Prices are US only Single spheroids are formed in the 384- and 96- well Nexcelom 3D Ultra- low attachment treated round bottom multi- well plates. Celigo image cytometer automatically monitors spheroid formation and growth tracking. U87 MG cells reproducibly form spheroids in Nexcelom 3D 384- and 96- well ultra- low attachment treated round bottom plates, as tracked by the Celigo image cytometer over time using bright- field imaging technique. 17- AAG inhibits the growth of U87 MG spheroids in Nexcelom 3D 384- well plates as detected and tracked over time using the Celigo image cytometer. Nexcelom 3D 384- well plates are used to detect U87 MG cells invading into the surrounding matrigel from spheroids over time. 17- AAG inhibits this invasion in a dosage dependent manner as recorded automatically by the Celigo image cytometer. References Advances in establishment and analysis of three- dimensional tumor spheroid- based functional assays for target validation and drug evaluation, Vinci, et al. (2012) BMC Biology 10: 29. Target Identification and Validation in Drug Discovery, Springer Protocols. Maria Vinci., A Tumor Spheroid- Based Migration Assays for Evaluation of Therapeutic Agents Vol. 986, p , 2013 Three- Dimensional (3D) Tumor Spheroid Invasion Assay. Maria Vinci, Carol Box, Suzanne A. Eccles. Jove. Issue 99,

2 Spheroid formation and growth inhibition using 96- and 384- well Nexcelom 3D round bottom ULA plates Key parameters from the spheroid formation and growth inhibition assay 384- well round bottom plate 96- well round bottom plate Cell type Glioblastoma U87 MG Glioblastoma U87 MG Seeding density (cells/well) Volume (µl) Spheroid formation time (days) 4 4 Replace portion of the media with 40 µl 100 µl 17- AAG Imaged by Celigo (t=0 from day 4) t= 0h, 24h, 48h, 72h t= 0h, 24h, 48h, 72h Imaging Bright- field Bright- field Calculate spheroid diameter Yes Yes Calculate estimated spheroid Yes Yes volume Obtain IC 50 Yes Yes Plate map for the 384- well round bottom plates 2

3 Spheroid growth and inhibition are monitored over time using Celigo Image Cytometer Bright- field image Day 4 Control 10uM 17AAG Visualized segmentation by Celigo Control 10uM 17AAG 17- AAG Dose (µm) Mean Spheroid Volume (µm 3 ) STD CV% N Control 3.99E E % E E % E E % E E % E E % E E % E E % E E % E E % 8 3

4 17- AAG inhibits the growth of the U87 MG spheroid over the time in a dose dependent manner using Nexcelom 3D 384- well plates Day 1 Day 4 Day 7 Day 14 Day 3 spheroid volume vs. 17- AAG concentration measured using Nexcelom 3D 96- well plates 2.E+08 d3 2.E+08 Volume (um3) 1.E+08 5.E+07 0.E+00 Control [17AAG] (µm)

5 Spheroid- based invasion assay using Nexcelom3D 384- well round bottom ULA plates Key parameters from the spheroid invasion assay Cell type 384- well round bottom plate Glioblastoma U87 MG Seeding density (cells/well) 500 Volume (µl) 80 Spheroid formation time (days) 4 Replace portion of the media with 1:1 matrigel and 17- AAG Imaged by Celigo (t=0 from day 4) 40 µl t= 0h, 24h, 48h, 72h # of image per well 1 Imaging Imaging analysis method Bright field Confluence using bright field image 1. U87 MG cells are plated at 500 cells/ well in 80 µl in the 384 well plates and allowed to grow to form spheroid for four days. 2. On day 4, 20 µl matrigel is added to 20 µl of media containing 17- AAG at the concentration of 10, 5, 2.5, 1.25, 0.625, , , , µm, and then added to each well as depicted in the plate map. 3. Plates are incubated at 37 o C, 5% CO 2 and imaged on day 4 (0hrs) and then at 24, 48 and 72 hrs 5

6 Invasion inhibition plate map for the 384- well round bottom plates Spheroid counting heat map per well 0hrs 24hrs 48hrs 72hrs The heat maps generated by Celigo software are used as a quality control tool to monitor the single spheroid status for each well. The results from the Nexcelom 3D invasion experiment demonstrated the uniformity of the invasion assay. 6

7 17- AAG inhibits the invasion of the U87 MG cells into matrigel over the time using Nexcelom 3D 384- well plates Control Bright- field image Day0 Day1 Day2 Day3 Visualized segmentation 10 µm 17- AAG Bright- field image Visualized segmentation by Celigo 7

8 17- AAG inhibits the invasion of the U87 MG cells into matrigel over the time in a dose dependent manner using Nexcelom 3D 384- well plates Inhibitory effect of 17- AAG on invasion of U87 MG cells invade into Matrigel in Nexcelom 3D multi- well plates. 8