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).

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