Overview

This protocol outlines a method for preparing samples with low cell numbers for transmission electron microscopy (TEM) analysis. It aims to provide efficient and reproducible results, revealing cellular structures in a physiological context.

Key Study Components

Area of Science

Cell Biology
Microscopy Techniques
Biomedical Research

Background

Transmission electron microscopy (TEM) is crucial for visualizing cellular structures.
Low cell numbers can pose challenges in sample preparation.
Efficient methods are needed to obtain reliable data from limited samples.
This protocol addresses these challenges by detailing a reproducible technique.

Purpose of Study

To prepare samples with low cell numbers for TEM analysis.
To enhance the understanding of cellular structures in biomedical research.
To provide a method that yields both qualitative and quantitative results.

Methods Used

Sorting of 10,000 hematopoietic stem cells (HSCs) into microcentrifuge tubes.
Using isocoves modified Dulbecco medium with 10% FBS for cell suspension.
Centrifugation at 1,000 times G for 10 minutes to concentrate cells.
Preparation for TEM analysis to visualize cellular structures.

Main Results

The method allows for effective sample preparation from low cell numbers.
Both qualitative and quantitative data can be obtained.
Reproducibility of results enhances the reliability of findings.
Cellular structures are clearly visualized under TEM.

Conclusions

This protocol is a valuable tool for researchers working with limited cell samples.
It supports the investigation of cellular structures in a physiological context.
The technique can be applied broadly in biomedical research.

Frequently Asked Questions

What is the main advantage of this TEM preparation method?

The main advantage is the ability to obtain reliable qualitative and quantitative results from low cell numbers.

How many cells are needed for this protocol?

The protocol requires sorting 10,000 hematopoietic stem cells.

What medium is used for cell suspension?

Isocoves modified Dulbecco medium with 10% FBS is used.

What is the centrifugation speed and time?

Centrifugation is performed at 1,000 times G for 10 minutes.

What type of microscopy is used in this study?

Transmission electron microscopy (TEM) is used to analyze the samples.

Can this method be applied to other cell types?

Yes, while this protocol focuses on HSCs, it can be adapted for other cell types.

Institution - Cincinnati Children’s Research Foundation

Articles Published in JoVE