Chemotaxis in neutrophil-like HL-60 cells - PubMed (original) (raw)

Chemotaxis in neutrophil-like HL-60 cells

Arthur Millius et al. Methods Mol Biol. 2009.

Abstract

Asymmetric localization of intracellular proteins and signals directs movement during axon guidance, endothelial cell invasion, and immune cell migration. In these processes, cell movement is guided by external chemical cues in a process known as chemotaxis. In particular, leukocyte migration in the innate immune system has been studied in the human neutrophil-like cell line (HL-60). Here, we describe the maintenance and transfection of HL-60 cells and explain how to analyze their behavior with two standard chemotactic assays. Finally, we demonstrate how to fix and stain the actin cytoskeleton of polarized cells for fluorescent microscopy imaging.

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Figures

Fig. 1

Fig. 1

Passaging and differentiating HL-60 cells. When cells reach a density between 1 and 2 million cells/ml, split to 0.15 million cells/ml in a total volume of 10 ml prewarmed culture medium. Differentiate cells in culture medium plus 1.3% DMSO; cells take ~5 days to become migratory.

Fig. 2

Fig. 2

Transient transfection of HL-60 cells with amaxa nucleofection. Spin ~5 million cells at 100 × g. Aspirate supernatant and resuspend pellet in 100 μl transfection solution per reaction and nucleofect with amaxa program “Y-001.” Flush with prewarmed recovery medium and incubate in an Eppendorf tube for 30 min. Transfer to a 6-well dish with 1.5 ml of recovery medium; expression occurs after 2 h. Shown is an example of HL-60 cells 5 h after transfection with GFP visualized with DIC and fluorescence microscopy.

Fig. 3

Fig. 3

Preparing a coverslip for live cell microscopy. (a) Dissolve 1 mg of bovine fibronectin in sterile water. After 1 h, add 4 ml of PBS and store 200 μg/ml fibronectin solution at 4°C. (b) Remove gaskets from plastic permanox 8-well chamber. Cut epoxy mold squares and stick to No. 1.5 gold seal cover glass. Add 125 μl of fibronectin, let sit for 1 h, rinse once with RPMI culture medium, and store in RPMI medium until ready to image.

Fig. 4

Fig. 4

An example of an HL-60 cell crawling toward a micropipette visualized with DIC and TIRF microscopy. Asterisk indicates micropipette tip.

Fig. 5

Fig. 5

The components of the EZ-TAXIS system are shown in (a) with the individual components in their order of assembly from top to bottom shown in (b). (c) An example of HL-60 cells migrating toward chemoattractant in the EZ-TAXIS assay visualized with brightfield microscopy.

Fig. 6

Fig. 6

Staining the actin cytoskeleton. Add 2× fixation buffer to plated cells and fix for 20 min at 4°C. Remove fixation buffer and replace with stain buffer for 20 min; protect from light. Shown is an example of an HL-60 cell stained with rhodamine phalloidin visualized with structured illumination microscopy.

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References

    1. Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, et al. Cell migration: integrating signals from front to back. Science. 2003;302:1704–1709. - PubMed
    1. Collins SJ, Gallo RC, Gallagher RE. Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture. Nature. 1977;270:347–349. - PubMed
    1. Collins SJ, Ruscetti FW, Gallagher RE, Gallo RC. Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc Natl Acad Sci USA. 1978;75:2458–2462. - PMC - PubMed
    1. Rao KM, Currie MS, Ruff JC, Cohen HJ. Lack of correlation between induction of chemotactic peptide receptors and stimulus-induced actin polymerization in HL-60 cells treated with dibutyryl cyclic adenosine monophosphate or retinoic acid. Cancer Res. 1988;48:6721–6726. - PubMed
    1. Carter BZ, Milella M, Tsao T, McQueen T, Schober WD, Hu W, et al. Regulation and targeting of antiapoptotic XIAP in acute myeloid leukemia. Leukemia. 2003;17:2081–2089. - PubMed

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