Spheroids that were previously stained with PI were cleared by using the ClearT solutions [19 (link),32 (link)] (Figure 1 ). Briefly, different formamide solutions (20, 40, 80 and 95% (v/v) in PBS) were freshly prepared. Then, spheroids were serially incubated with 20, 40, 80 and 95% formamide solutions during 5 min each, and lastly with 95% formamide solution during 15 min. To assess the best conditions for spheroids’ clearing, spheroids were immersed in the clearing solutions under three different conditions: (i) static conditions where no agitation was used—ClearT/S; (ii) horizontal agitation at 200 RPM by using an orbital mixer (LBX MM1500 series, Labbox, Barcelona, Spain)—ClearT/H; and (iii) rotatory agitation using a tube roller shaker (MX-T6-S Analog Tube Roller, Scilogex, city, CT, USA) at 50 RPM—ClearT/R. All the methods were performed at room temperature (RT). After the clearing, spheroids were transferred to μ-slide 8 well imaging plates (Ibidi GmbH) to be imaged by CLSM (Section 2.2.6 ).
μ slide 8 well imaging plates
Manufactured by Ibidi
Sourced in Germany
The μ-Slide 8 Well is an imaging plate designed for live-cell microscopy. It features eight individual wells, allowing for the simultaneous observation and analysis of multiple samples. The plate is made of high-quality materials and is optimized for consistent imaging performance.
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5 protocols using μ slide 8 well imaging plates
1
Optimizing Spheroid Clearing Using Clear^T
2
Clearing PI-stained Spheroids using ClearT2
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PI-stained spheroids were cleared by using ClearT2 solutions [21 (link),32 (link)]—Figure 1 . Initially, 25% formamide/10% PEG 4000 Da and 50% formamide/20% PEG 4000 Da solutions were freshly prepared, as previously described in detail by Kuwajima [32 (link)]. Then, spheroids were incubated with 25% formamide/10% PEG solution during 10 min, 50% formamide/20% PEG for 5 min and lastly 50% formamide/20% PEG for 1 h. During the clearing procedure, three different conditions were tested, as described in Section 2.2.3 for the ClearT method: (i) ClearT2/S; (ii) ClearT2/H; and (iii) ClearT2/R. All methods were performed at RT. After the clearing, spheroids were transferred to μ-slide 8 well imaging plates (Ibidi GmbH) to be imaged via CLSM (Section 2.2.6 ).
3
Clearing Spheroids with PEG Solutions
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The Clear T2 clearing method variations (Clear T2 ∕4, Clear T2 ∕8, and Clear T2 ∕10 that use PEG with an MW of 4000, 8000, and 10,000 Da, respectively) were performed accordingly to previous works (see Fig. 1 for the pipeline overview of the method). 22, (link)26 (link) Initially, whole PI-stained spheroids were immersed in a 25% formamide/10% PEG solution for 10 min. Afterward, spheroids were immersed in 50% formamide/20% PEG solution for 5 min and last in another 50% formamide/20% PEG solution for 1 h. PEG 4000, 8000, and 10,000 Da were used to prepare the solutions used in the Clear T2 ∕4, Clear T2 ∕8, and Clear T2 ∕10 method, respectively. All Clear T2 methods were performed at room temperature using a plate shaker at 100 RPM.
For comparative purposes, some spheroids were only immersed in PBS instead of the clearing solutions (noncleared PI-stained spheroids). Moreover, nonstained spheroids were subjected to Clear T2 methods (cleared nonstained spheroids) to evaluate the influence of the method in the spheroids' autofluorescence.
After the clearing of the intact spheroids, samples were transferred to μ-slide 8-well imaging plates (Ibidi GmbH, Germany) for imaging experiments.
For comparative purposes, some spheroids were only immersed in PBS instead of the clearing solutions (noncleared PI-stained spheroids). Moreover, nonstained spheroids were subjected to Clear T2 methods (cleared nonstained spheroids) to evaluate the influence of the method in the spheroids' autofluorescence.
After the clearing of the intact spheroids, samples were transferred to μ-slide 8-well imaging plates (Ibidi GmbH, Germany) for imaging experiments.
4
Hydrogel Cell Visualization via CLSM
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Confocal laser scanning microscopy (CLSM) was used to visualize the cell distribution within hydrogels. Cells (1.6 × 104 cells/well) were seeded in hydrogels in μ-Slide 8-well Ibidi imaging plates (Ibidi GmbH, Planegg/Martinsried, Germany) on the surface of the hydrogel. After 24 h and 72 h, the nucleus of the cells were stained with Hoechst 33342 (2 μM, Thermo Fisher Scientific, Waltham, MA, USA), whereas the hydrogels were labelled with calcein (40 μg/mL). Then, the imaging experiments were performed using a Zeiss LSM710 laser scanning confocal microscope (Carl Zeiss AG, Oberkochen, Germany), where consecutive z-stacks were acquired. The 3D reconstruction and image analysis were performed using Zeiss Zen 2010 software [33 (link)].
5
CLSM Visualization of Cell Distribution in Hydrogels
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Confocal laser scanning microscopy (CLSM) was used to visualize the cell distribution within hydrogels. HUVEC Cells (1.6 × 104 cells/well) were seeded in hydrogels in μ-Slide 8-well Ibidi imaging plates (Ibidi GmbH, Planegg/Martinsried, Germany) on the surface of the hydrogel. After 48 h, the nucleus of the cells were stained with Hoechst 33342 (2 μM, Thermo Fisher Scientific, Waltham, MA, USA). Then, the imaging experiments were performed, using a Zeiss LSM710 laser scanning confocal microscope (Carl Zeiss AG, Oberkochen, Germany), whereupon consecutive z-stacks were acquired. The 3D reconstruction and image analysis were performed using Zeiss Zen 2010 software.
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