Needle Assay for Chemotaxis Dynamics

A needle assay was performed as described (Iijima and Devreotes, 2002 (link); Zhang et al., 2010 (link); Wang et al., 2011b (link); Chen et al., 2012 (link)). Cells were cultured in HL5 medium, washed twice with DB, and shaken for 1 h before being induced to differentiate with 100 nM cAMP pulses at 6-min intervals for 4 h. Cells were then plated on a chambered coverslip (Lab-Tek; Nalgen Nunc, Rochester, NY). A cAMP gradient was produced by a micropipette (Femtotips; Eppendorf, Hamburg, Germany) containing 1 μM cAMP and a microinjector with a compensation pressure of 100 hPa (FemtoJet; Eppendorf). Images of moving cells were recorded at 30-s intervals for 20 min using a DMI6000 (Leica) and a Yokogawa CSU10 spinning-disk confocal microscope equipped with a 10× objective connected to a digital camera. ImageJ software (National Institutes of Health, Bethesda, MD) was used to analyze data.

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Senoo H., Cai H., Wang Y., Sesaki H, & Iijima M. (2016). The novel RacE-binding protein GflB sharpens Ras activity at the leading edge of migrating cells. Molecular Biology of the Cell, 27(10), 1596-1605.

Publication 2016

Femtotips FemtoJet DMI6000 CSU10

Assay Camera Cells Confocal microscope Cultured medium Needle Pressure Pulses

Corresponding Organization : Johns Hopkins University

Other organizations : Chinese Academy of Sciences, Institute of Biophysics

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Variable analysis

independent variables

Induction of differentiation with 100 nM cAMP pulses at 6-min intervals for 4 h

dependent variables

Cell movement in response to a cAMP gradient

control variables

Cell culture in HL5 medium
Washing cells twice with DB
Shaking cells for 1 h before induction
Plating cells on a chambered coverslip
Producing a cAMP gradient using a micropipette and microinjector

controls

No positive or negative controls were explicitly mentioned in the protocol.

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