A Convenient Technique to Fix Suspension Cells on a Coverslip for Microscopy

Keiko Mihara1, Tomofumi Nakayama1, Hisato Saitoh2

1 These authors contributed equally to this work, 2 Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.30
DOI:  10.1002/0471143030.cb0430s68
Online Posting Date:  September, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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. © 2015 by John Wiley & Sons, Inc.

Keywords: myeloid cell; suspension cell culture; cell adhesion; cell fixation

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Visualization of DNA in the Nucleus and Actin Fibers in the Cytoplasm of HL‐60 Cells
  • Support Protocol 1: Conditions for ATRA Exposure for In Vitro Differentiation of HL‐60 Cells to Neutrophil‐Like Cells
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Visualization of DNA in the Nucleus and Actin Fibers in the Cytoplasm of HL‐60 Cells

  Materials
  • HL‐60 cells (e.g., American Type Culture Collection [ATCC], cat. no. CCL‐240) or ATRA‐induced dHL‐60 (see protocol 2Support Protocol)
  • RPMI‐1640 with L‐glutamine and phenol red medium (e.g., Wako Pure Chemical Industries, cat. no. 189‐02025)
  • RPMI‐1640 medium supplemented with 5% fetal bovine serum (FBS), 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate (e.g., Wako Pure Chemical Industries, cat. no. 168‐23191)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 4% paraformaldehyde‐PBS solution (see recipe)
  • Immunofluorescence (IF) solution (PBS containing 0.2% BSA and 0.1% Triton X‐100; see recipe)
  • Fluorescein phalloidin solution (see recipe)
  • 4′,6‐Diamidino‐2‐phenylindole (DAPI) solution (see recipe)
  • 1,4‐Diazabicyclo[2.2.2]octane (DABCO) solution (see recipe)
  • Culture dish, 10 × 35 mm (TrueLine, cat. no. TR4000)
  • 50‐ml disposable conical tubes (e.g., BD Falcon)
  • Circular coverslips, 12 mm in diameter (e.g., Fisher Scientific, cat. no. 12‐545‐80)
  • Forceps
  • Nail polish
  • Kimwipes
  • Paraffin film
  • Gilson Pipetman pipet (P1000)

Support Protocol 1: Conditions for ATRA Exposure for In Vitro Differentiation of HL‐60 Cells to Neutrophil‐Like Cells

  Additional Materials (also see protocol 1Basic Protocol)
  • HL‐60 cells (e.g., American Type Culture Collection [ATCC], cat. no. CCL‐240)
  • 1 mM all‐trans‐retinoic acid (ATRA) solution (see recipe)
  • Culture dish (20 × 100 mm)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Collins, S.J. 1987. The HL‐60 promyelocytic leukemia cell line: Growth, differentiation and cellular oncogene expression. Blood 70:1233‐1244.
  Hepper, I., Schymeinsky, J., Weckbach, L.T., Jakob, S.M., Frommhold, D., Sixt, M., Laschinger, M., Sperandio, M., and Walzog, B. 2012. The mammalian actin‐binding protein 1 is critical for spreading and intraluminal crawling of neutrophils under flow conditions. J. Immunol. 188:4590‐4601.
  Koh, C.M. 2013a. Preparation of cells for microscopy using cytospin. Methods Enzymol. 533:235‐240.
  Koh, C.M. 2013b. Preparation of cells for microscopy using chamber slides and coverslips. Methods Enzymol. 533:241‐247.
  Nakayama, T., Mihara, K., Kawata, J., Kimura, H., and Saitoh, H. 2014. Adhesion of suspension cells on a coverslip in serum‐free conditions. Anal. Biochem. 466:1‐3.
  Olins, A.L. and Olins, D.E. 2001. Cytoskeletal influences on nuclear shape in granulocytic HL‐60 cells. BMC Cell Biol. 5:30.
  Willingham, M.C. 2010. Fluorescence labeling of intracellular antigens of attached or suspended tissue‐culture cells. Methods Mol. Biol. 588:153‐164.
  Zuchero, J.B., Coutts, A.S., Quinlan, M.E., Thangue, N.B., and Mullins, R.D. 2009. p53‐cofactor JMY is a multifunctional actin nucleation factor. Nat. Cell Biol. 11:451‐459.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library