TITLE: Purification of Cyclic GMP-AMP from Viruses and Measurement of Its Activity in Cell Culture
AUTHORS: Alice Mayer, Jonathan Maelfait, Anne Bridgeman, Jan Rehwinkel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Sensing of cytoplasmic DNA by cGAS is essential for the initiation of immune responses against several viruses. cGAS also plays important roles in some autoinflammatory and autoimmune diseases and may be involved in immune responses targeting cancer cells. Once activated, cGAS catalyzes the formation of the di-nucleotide 2′-3′-cyclic GMP-AMP (cGAMP), which propagates a signaling cascade leading to the production of type I interferons (IFNs). Interestingly, cGAMP is incorporated into enveloped viruses and is transferred to newly infected cells by virions. In this article, we describe a method to purify cGAMP from viral particles and a bioassay to measure its activity. This assay takes advantage of a reporter cell line that expresses the genes encoding green fluorescent protein (GFP) and firefly luciferase under the control of the IFNß promoter, allowing the testing of several samples in a single experiment taking not more than 3 days.</div></div>

[STEPS]
?. [3.1 cGAMP Purification from Viral Particles]
Resuspend pelleted viruses in , transfer to 1.5 mL tubes, and incubate , vortex regularly.
[X-100 lysis buffer]
on ice
We typically analyze pelleted virus stocks corresponding to at least 106infectious units, although this will depend on the type of virus and the amount of cGAMP incorporated during budding. For highly concentrated samples, it is advisable to increase the volume of X-100 lysis buffer, in which case the volumes of P:I:C and chloroform need to be adjusted accordingly. It is noteworthy that we have not been able to detect cGAMP activity in extracts when using this protocol to recover cGAMP from DNA-stimulated cells. Nevertheless, it may be possible to use this method to purify cGAMP from cells overexpressing cGAS [16].
?. [3.1 cGAMP Purification from Viral Particles]
Centrifuge: .
Centrifuge: 1000 34, 10 min, 4 10
?. [3.1 cGAMP Purification from Viral Particles]
Optional: Spike  into  as a positive control (to test the efficiency of the purification process).
[cGAMP]
[X-100 lysis buffer]
?. [3.1 cGAMP Purification from Viral Particles]
Collect the supernatant, add  and incubate for .
[of benzonase]
on ice
?. [3.1 cGAMP Purification from Viral Particles]
Add , vortex vigorously, spin: .
[P:I:C]
Centrifuge: 17000 34, 5 min
All the steps involving P:I:C and chloroform should be performed in a chemical hood. These reagents, and also the tubes and pipette tips that have been in contact with them, should be disposed of in an appropriate way. Phase lock tubes can be used for these extractions.
?. [3.1 cGAMP Purification from Viral Particles]
Take off upper aqueous layer by pipetting carefully without disturbing the lower layer.
?. [3.1 cGAMP Purification from Viral Particles]
Add , vortex vigorously, spin: .
[P:I:C]
Centrifuge: 17000 34, 5 min
?. [3.1 cGAMP Purification from Viral Particles]
Take off upper aqueous layer by pipetting carefully without disturbing the lower layer.
?. [3.1 cGAMP Purification from Viral Particles]
Add , vortex vigorously, spin: .
[chloroform]
Centrifuge: 17000 34, 5 min
?. [3.1 cGAMP Purification from Viral Particles]
Transfer the upper aqueous layer onto Amicon 3 K filter column and centrifuge .
Centrifuge: 14000 34, 30 s
?. [3.1 cGAMP Purification from Viral Particles]
Dry the samples using a Speed Vac, resuspend pellets in , and store at .
[H2O]
-80 °C
?. [3.2.1 Seeding and Differentiation of THP-1 Reporter Cells (Day 0 of the Bioassay)]
50,000 p125-THP1 cells are seeded per well in the presence of .
[PMA]
The number of wells to seed depends on the number of samples, the number of dilutions of each sample, and the number of technical replicates (ideally triplicates). The number of dilutions depends on the expected concentration of cGAMP in the sample, taking into account that the assay usually saturates around 10 or 20 ng/well. We typically analyze between 4 and 6 dilutions per sample. Ideally, 30 wells for the standard (9 dilutions and a blank, all in triplicate) and 18 wells per sample are required
?. [3.2.1 Seeding and Differentiation of THP-1 Reporter Cells (Day 0 of the Bioassay)]
Harvest the THP-1 reporter cells, centrifuge, resuspend in R10 media, and count.
?. [3.2.1 Seeding and Differentiation of THP-1 Reporter Cells (Day 0 of the Bioassay)]
Adjust the cell concentration to 5 × 105 cells per mL.
?. [3.2.1 Seeding and Differentiation of THP-1 Reporter Cells (Day 0 of the Bioassay)]
Add PMA to a final concentration of .
?. [3.2.1 Seeding and Differentiation of THP-1 Reporter Cells (Day 0 of the Bioassay)]
Dispatch  per well in a flat-bottom 96-well plate.
100 µl
?. [3.2.1 Seeding and Differentiation of THP-1 Reporter Cells (Day 0 of the Bioassay)]
Place in a tissue culture incubator (, 5% CO2) and incubate .
37 °C
The cells should be incubated with PMA for at least 18 h, and this incubation can be extended up to 24 h. Shorter and longer incubation periods have not been tested with the luciferase readout; however, using flow cytometry, 12 h or 42 h incubation gave rise to a diminished fraction of GFP-positive cells.
?. [3.2.2 Stimulation of THP-1 Reporter Cells (Day 1 of the Bioassay)]
Warm some R10 media to  and bring nuclease-free distilled water to .
37 °C
0 Room temperature
?. [3.2.2 Stimulation of THP-1 Reporter Cells (Day 1 of the Bioassay)]
Thaw the 2× PERM buffer, the samples containing the cGAMP to dose and some cGAMP for the standard.
?. [3.2.2 Stimulation of THP-1 Reporter Cells (Day 1 of the Bioassay)]
Dilute the cGAMP-containing samples in nuclease-free distilled water to a total of  for duplicate measurements or  for triplicates.
55 µl
80 µl
We typically prepare the dilutions of the standard and the samples in sterile v-bottom 96-well plates, and then transfer these to the cells with a multichannel pipette.
We typically use only half of our samples (10 μL). If the sample contains a concentration of cGAMP very close to the lower detection limit, we suggest using all of it without serial dilution. In this case, add 7.5 μL of water to the 20 μL of sample and do the experiment in duplicate.
?. [3.2.2 Stimulation of THP-1 Reporter Cells (Day 1 of the Bioassay)]
Further dilute samples 1:2 with 2× PERM buffer, then titer down in twofold dilution series in 1× PERM buffer. We usually do between 4 and 6 dilutions per sample.
The number of wells to seed depends on the number of samples, the number of dilutions of each sample, and the number of technical replicates (ideally triplicates). The number of dilutions depends on the expected concentration of cGAMP in the sample, taking into account that the assay usually saturates around 10 or 20 ng/well. We typically analyze between 4 and 6 dilutions per sample. Ideally, 30 wells for the standard (9 dilutions and a blank, all in triplicate) and 18 wells per sample are required.
?. [3.2.2 Stimulation of THP-1 Reporter Cells (Day 1 of the Bioassay)]
Dilute the (2′-3′) cGAMP standard in 1× PERM buffer, from 50 ng/well to 0.02 ng/well in two-fold dilution series. Prepare  of each dilution (triplicates). Do not forget to keep  as a negative control (blank).
80 µl
[1× PERM buffer only]
?. [3.2.2 Stimulation of THP-1 Reporter Cells (Day 1 of the Bioassay)]
Wash the reporter cells by removing the medium and by replacing it with .
[fresh R10]
In all the wash steps, media can be removed with a multichannel pipette or flicked off the plate.
?. [3.2.2 Stimulation of THP-1 Reporter Cells (Day 1 of the Bioassay)]
Remove all medium.
?. [3.2.2 Stimulation of THP-1 Reporter Cells (Day 1 of the Bioassay)]
Gently overlay the cells with .
[sample or standard dilutions]
?. [3.2.2 Stimulation of THP-1 Reporter Cells (Day 1 of the Bioassay)]
Incubate for  in a tissue culture incubator.
?. [3.2.2 Stimulation of THP-1 Reporter Cells (Day 1 of the Bioassay)]
Wash the cells by adding  per well, and then remove all medium.
[fresh R10]
?. [3.2.2 Stimulation of THP-1 Reporter Cells (Day 1 of the Bioassay)]
Add  and incubate between  and  in a tissue culture incubator.
[fresh R10 media]
?. [3.2.3 Luciferase Assay (Day 2 of the Bioassay)]
The activation of the IFNß promoter can also be assessed by flow cytometry. This method takes more time, but is an alternative if you do not have access to the equipment needed for luciferase assays. In that case, the cells should be harvested 6 h after stimulation as follows: (1) flick off the supernatant and add 200 μL of ice-cold FACS buffer (PBS, 2 mM EDTA, 1% FCS, and 0.02% sodium azide), (2) incubate for a minimum of 2 minutes on ice, (3) detach the cells by pipetting up and down and transfer to a v-bottom 96-well plate, (4) centrifuge 5 min at 500 × g, (5) flick off the supernatant and resuspend the cells in 100 μL of FACS buffer containing 1 ng/mL of DAPI. The fold increase of the GFP median fluorescence intensity (gated on DAPI-negative cells) is comparable to the luciferase assay.
?. [3.2.3 Luciferase Assay (Day 2 of the Bioassay)]
Dilute the One Glo reagent 1:2 with R10 media, protect from light, and wait until it reaches .
0 Room temperature
?. [3.2.3 Luciferase Assay (Day 2 of the Bioassay)]
Flick off the media from the plates containing the THP1 cells and replace with .
[diluted One Glo reagent]
?. [3.2.3 Luciferase Assay (Day 2 of the Bioassay)]
Incubate for  at  in the dark.
0 Room temperature
?. [3.2.3 Luciferase Assay (Day 2 of the Bioassay)]
Pipette up and down to homogenize and transfer  to a white 96-well plate.
75 µl
?. [3.2.3 Luciferase Assay (Day 2 of the Bioassay)]
Read with a luminometer according to the manufacturer’s instructions.