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A Convenient Technique to Fix Suspension Cells on a Coverslip for Microscopy
Keiko Mihara1,2, Tomofumi Nakayama1,2, Hisato Saitoh1
1Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto, Japan.
Abstract:
Human myeloid HL-60 cells are usually cultured in suspension in medium containing 5% to 10% fetal bovine serum (FBS) and thus are often difficult to adhere to a coverslip. In this unit, we describe how removal of FBS from the culture medium facilitates adhesion of HL-60 cells to coverslips. Importantly, HL-60 cells that adhere to the coverslips immersed in FBS-free medium can be immobilized in situ by conventional chemical fixatives and thus permeabilized for probing cellular structures using specific dyes and/or reagents, followed by microscopic observation. All-trans-retinoic-acid-exposed differentiated HL-60 cells, which have properties similar to neutrophils, can also adhere efficiently to coverslips in FBS-free medium. Because the procedure is not complex and special equipment is not required, the simplicity and cost effectiveness of this FBS-free cell adhesion protocol may be beneficial to researchers who are interested in assessing the structure and function of suspension cells using microscopy.
Insights
Removing fetal bovine serum (FBS) from culture medium allows human myeloid HL-60 cells to adhere to coverslips. This simplified method enables microscopic analysis of cell structure and function, proving cost-effective for researchers.
Area of Science:
- Cell Biology
- Microscopy Techniques
Background:
- Human myeloid HL-60 cells are typically suspension-cultured with fetal bovine serum (FBS), hindering their adhesion to coverslips for microscopic analysis.
- Assessing the structure and function of suspension cells often requires specialized techniques due to poor adherence.
Purpose of the Study:
- To develop a simple and cost-effective method for improving the adherence of HL-60 cells to coverslips.
- To enable detailed microscopic observation of HL-60 cell structures and functions.
Main Methods:
- Culturing HL-60 cells in a serum-free medium to promote coverslip adhesion.
- Immobilizing adhered cells using standard chemical fixatives for permeabilization.
- Utilizing specific dyes and reagents for microscopic examination.
Main Results:
- Removal of FBS significantly enhanced the adhesion of HL-60 cells to coverslips.
- Differentiated HL-60 cells, mimicking neutrophil properties, also adhered effectively in FBS-free medium.
- The protocol allows for in situ fixation and permeabilization for detailed cellular probing.
Conclusions:
- A simplified, FBS-free cell adhesion protocol effectively facilitates HL-60 cell adherence for microscopy.
- This cost-effective method is suitable for researchers studying suspension cell structure and function.
- The technique is adaptable for differentiated HL-60 cells with neutrophil-like characteristics.
