Oct 14, 2019

Public workspaceStaining cells with IncuCyte Cytolight Rapid Dyes for flow cytometry or fluorescent microscopy

DOI
dx.doi.org/10.17504/protocols.io.73nhqme
  • 1Medical University of South Carolina
  • Hammer Lab
    Tech. support phone: +18437924527 email: arman@hammerlab.org
Elinor Gottschalk
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DOI: dx.doi.org/10.17504/protocols.io.73nhqme
Protocol Citation: Elinor Gottschalk, Bulent Arman Aksoy, Pinar Aksoy, Eric Czech, Jeff Hammerbacher 2019. Staining cells with IncuCyte Cytolight Rapid Dyes for flow cytometry or fluorescent microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.73nhqme
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 09, 2019
Last Modified: October 14, 2019
Protocol Integer ID: 28494
Abstract
This protocol is for labeling cells with IncuCyte Cytolight Rapid dyes from Essen Bioscience. We optimized the dye concentration for Jurkat cells, pmel-1 T cells, and B16F10 melanoma cells.
Guidelines
We optimized IncuCyte® CytoLight Rapid Green Reagent for live cell cytoplasmic labeling with Jurkat cells, pmel-1 T cells, and IncuCyte® CytoLight Rapid Red Reagent with B16F10 melanoma cells. The optimal concentrations (based on minimal decrease in cell viability and highest percentage of stained cells) were:
  • Jurkat: 1 µM (green)
  • pmel-1 T cells: 0.11 µM (green)
  • B16F10 cells: 0.11 µM (red)

We visualized the stained cells by flow cytometry or on the Keyence BZ-X710 fluorescence microscope equipped with GFP and Cy5 filters.


Materials
MATERIALS
ReagentIncuCyte® CytoLight Rapid Green Reagent for live cell cytoplasmic labelingEssen BiosciencesCatalog #4705
Reagent1X PBSVWR International (Avantor)Catalog #75800-986
ReagentIncuCyte® CytoLight Rapid Red Reagent for live cell cytoplasmic labelingEssen BiosciencesCatalog #4706
Harvest cells and wash with 1X PBS
Centrifuge Centrifigation350 x g , 5 minutes
Resuspend cells in PBS at 1 million cells per ml
Add IncuCyte Dye
  • For pmel-1 T cells: we determined that 0.11 µM of green dye was optimal
  • We made our working stock 11 µM (so for every 1 ml of cell suspension, we add 10 ul of working stock of dye)
  • For Jurkat cells, 1 µM of green dye was optimal
  • For B16F10 cells, 0.11 µM of red dye was optimal
Incubate the cells at Temperature37 °C for Duration00:20:00 (we wrapped the tube in foil and placed it in a water bath).
  • invert the tube twice during incubation to mix the cells
Add a 6-fold volume of complete media (containing serum) to bind the excess dye.
Centrifuge Centrifigation350 x g , 5 minutes
Resuspend cell in complete media at the desired concentration.
  • Two different cell types can be labeled with different dyes and then co-cultured and visualized
  • Stained T cells can be visualized migrating in collagen gel
Assay the cells via flowcytometry or fluorescence microscopy.
Flow cytometry: FITC for green dye; APC for red dye
Fluorescence microscopy: FITC filter for green dye; Cy5 filter for red dye