DC
Darran G. Cronshaw
  • Center for Autoimmune and Musculoskeletal Diseases, Feinstein Institute for Medical Research, USA
Research fields
  • Immunology
Transfection of S2 Cell with DNA Using CellFectin Reagent
Author:  Darran G. Cronshaw, date: 03/05/2012, view: 22162, Q&A: 0
This method provides a step-by-step guide to transfecting Drosophila S2 cells with the pRmHA-3 (or similar) vector with insert of choice (in this case SDF-1β-FLAG) and generating a stable cell line. This cell line is then capable of producing the protein of interest under inducible conditions by addition of copper sulfate, which can then be purified and used as desired. This protocol provides an example to finding out when your peak protein production occurs and a method for determining optimal selection conditions.
Calcium Mobilisation Assay in Response to Chemokine Stimulation
Author:  Darran G. Cronshaw, date: 05/05/2012, view: 14862, Q&A: 1
This assay is used to measure calcium mobilization in lymphocytes (either in primary cells or cell lines) in response to chemokine stimulation using ratiometric analysis. It has also been used for measuring TCR mediated calcium flux. In addition, the same labeling procedure with the addition of brilliant black (a quenching agent) (Sigma-Aldrich, catalog number: 211842) in the loading buffer (at 100 μM) allows for quantification using the FLIPR system on poly-D-lysine plates. Probenicid is an anion-exchange protein inhibitor and prevents the extrusion of the dyes by organic ion transporters.
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