Embedding 3D Spheroids in Agarose Gel Drops: A Technique to Preserve Spheroids for Downstream Analysis
Published: April 30, 2023
Abstract
Source: Rolver, M. G. et al. Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells. J. Vis. Exp. (2019)
This video describes the technique to embed 3D spheroids in agarose gel drops for preservation. The embedded 3D spheroids can be further used for downstream analysis.
Protocol
1. Embedding of 3D Spheroids Prepare the agarose gel into which the spheroids are embedded (only necessary first time performing the protocol) Mix 1 g of bacto-agar in 50 mL of ddH₂O. Heat slowly in the microwave oven until the bacto-agar has dissolved and a homogeneous gel has formed. Do not allow the gel to boil. Keep the bacto-agar warm in a water bath at 60 °C. Keep at 4 °C between experiments. Embedding …
Representative Results
Figure 1: Fixing, embedding and immunohistochemistry analysis of spheroids. (A) Schematic representation of the protocol for embedding of spheroids. Individual steps are marked as (i-vii). (B) Image of embedded MDA-MB-231 spheroid. Scale bar: 50 µm. (C) Representative image of chemotherapy-treated MDA-MB-231 sphe…
Divulgaciones
The authors have nothing to disclose.
Materials
| Bactoagar | BD Bioscience | #214010 | Used for agarose gel preparation |
| Formaldehyde | VWR Chemicals | #9713.1000 | Used for cell fixation |
| Medim Uni-safe casette | Medim Histotechnologie | 10-0114 | Used for storage of embedded spheroids |
| Superfrost Ultra-Plus Adhesion slide | Menzel-Gläser | #J3800AMNZ | Microscope glass slide used for embedding |
| CellTiter-Glo 3D Cell Viability Assay | Promega | #G9681 | Used for the cell viability assay |
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Embedding 3D Spheroids in Agarose Gel Drops: A Technique to Preserve Spheroids for Downstream Analysis. J. Vis. Exp. (Pending Publication), e20481, doi: (2023).