14. Concanavalin A (ConA) stock: 10 mg/mL in sterile DDW
(10Â stock). Store 500 μL aliquots at À20
C.
15. ConA-coated plate activation solution: 50 mM CaCl 2 , 50 mM
MnCl 2 in DDW. Filter sterilize, store at room temperature.
16. Fluorescent wide-field microscope of choice for live-cell image
acquisition (see Note 1).
17. Image analysis software: FISH-quant [82], free software developed in the MATLAB programming language (MathWorks).
Download the FISH-quant package (http://code.google.
com/p/fish-quant/) together with the MCRInstaller, which
allows one to run a MATLAB algorithm without separately
installing MATLAB onto the computer.
18. Image analysis software: Fiji (Java software for imageprocessing analysis; freely available at https://fiji.sc/).
19. Image analysis software: CellProfiler [83], for cell outline generation (freely available at https://cellprofiler.org/).
20. Image analysis software: For image deconvolution use a software such as the Huygens Software Suite (https://svi.nl/
HomePage).
2.2 Materials
for Visualizing
MS2-Labeled RNAs
in Mammalian Cells,
with a Focus
on Analyzing
Transcription
Dynamics
1. Transcription reporter constructs containing MS2 stem-loop
repeats: pIntro-MS2x64 and pIntro-MS2x128 [60]. These
plasmids are available upon request (see Fig. 4).
2. Lentiviral plasmid for MCP fused to a fluorescent protein:
pHAGE-Ubc-NLS-MCP-GFP (available upon request) or
pHAGE-Ubc-NLS-tdMCP-GFP (Addgene #40649).
3. Packaging plasmids for lentivirus production (available upon
request): pHDM-tat1b (helper plasmid for lentiviral vector,
HIV tat driven by CMV promoter); pRC-CMV-rev1b (helper
plasmid for lentiviral vector, rev1b driven by CMV promoter);
pHDM-Hgpm2 (helper plasmid for lentiviral vector, has
codon-optimized HIV gag-pol driven by CMV promoter);
pHDM-G (helper plasmid for lentiviral vectors, VSV-G driven
by CMV promoter).
4. XL1-Blue competent cells (see Note 2).
5. HeLa Flp-in H9 cell line [60], allowing the generation of
isogenic stable cell lines by genomic integration in a single
integrated Flp recombination target (FRT) site from pFRT/
Lac Zeo, under zeocin selection (100 μg/mL) (see Notes 3
and 4).
6. DMEM + GlutaMAX supplemented with 10% fetal bovine
serum (FBS) and penicillin/streptomycin (P/S; 10 U/mL),
in a humidified CO2 incubator at 37
C for propagation
conditions.
Imaging Single mRNAs in Living Eukaryotic Cells
131
(10Â stock). Store 500 μL aliquots at À20
C.
15. ConA-coated plate activation solution: 50 mM CaCl 2 , 50 mM
MnCl 2 in DDW. Filter sterilize, store at room temperature.
16. Fluorescent wide-field microscope of choice for live-cell image
acquisition (see Note 1).
17. Image analysis software: FISH-quant [82], free software developed in the MATLAB programming language (MathWorks).
Download the FISH-quant package (http://code.google.
com/p/fish-quant/) together with the MCRInstaller, which
allows one to run a MATLAB algorithm without separately
installing MATLAB onto the computer.
18. Image analysis software: Fiji (Java software for imageprocessing analysis; freely available at https://fiji.sc/).
19. Image analysis software: CellProfiler [83], for cell outline generation (freely available at https://cellprofiler.org/).
20. Image analysis software: For image deconvolution use a software such as the Huygens Software Suite (https://svi.nl/
HomePage).
2.2 Materials
for Visualizing
MS2-Labeled RNAs
in Mammalian Cells,
with a Focus
on Analyzing
Transcription
Dynamics
1. Transcription reporter constructs containing MS2 stem-loop
repeats: pIntro-MS2x64 and pIntro-MS2x128 [60]. These
plasmids are available upon request (see Fig. 4).
2. Lentiviral plasmid for MCP fused to a fluorescent protein:
pHAGE-Ubc-NLS-MCP-GFP (available upon request) or
pHAGE-Ubc-NLS-tdMCP-GFP (Addgene #40649).
3. Packaging plasmids for lentivirus production (available upon
request): pHDM-tat1b (helper plasmid for lentiviral vector,
HIV tat driven by CMV promoter); pRC-CMV-rev1b (helper
plasmid for lentiviral vector, rev1b driven by CMV promoter);
pHDM-Hgpm2 (helper plasmid for lentiviral vector, has
codon-optimized HIV gag-pol driven by CMV promoter);
pHDM-G (helper plasmid for lentiviral vectors, VSV-G driven
by CMV promoter).
4. XL1-Blue competent cells (see Note 2).
5. HeLa Flp-in H9 cell line [60], allowing the generation of
isogenic stable cell lines by genomic integration in a single
integrated Flp recombination target (FRT) site from pFRT/
Lac Zeo, under zeocin selection (100 μg/mL) (see Notes 3
and 4).
6. DMEM + GlutaMAX supplemented with 10% fetal bovine
serum (FBS) and penicillin/streptomycin (P/S; 10 U/mL),
in a humidified CO2 incubator at 37
C for propagation
conditions.
Imaging Single mRNAs in Living Eukaryotic Cells
131