Sorted cells were pelleted and resuspended in 275uL of 1x dsDNase buffer (Thermofisher#EN0771) and incubated for 15 minutes after which 25uL of dsDNase enzyme (Thermofisher#EN0771) was added and the sample was incubated at 37°C for 2 hours. After incubation, 3uL of 1M DTT was added to the sample to quench dsDNAse activity and incubated at 55°C for 5 minutes for heat inactivation. Samples were pelleted at 850xg for 5 minutes, resuspended in 500uL of pre-warmed wash buffer, and incubated for 10 minutes. This step was repeated once for a total of 2 washes. Samples were then resuspended in 500uL of SSCT buffer and incubated for 5 minutes. Samples were centrifuged and resuspended in 1mL of 0.5x PBS and 0.02% BSA (Thermofisher #AM2616) and 0.2u/uL RNAse inhibitor (Millipore Sigma #3335399001). Polymer Disassembly was assessed by measurement of fluorescence intensity on the BD FACSAria III.
En0771
Manufactured by Thermo Fisher Scientific
The EN0771 is a laboratory centrifuge designed for general-purpose use. It is capable of separating samples based on their density and size. The centrifuge can accommodate a variety of sample tubes and microplates. Technical specifications and performance details are available upon request.
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Lab products found in correlation
5 protocols using en0771
1
Polymer Disassembly Evaluation by FACS
2
Selective RNA/DNA Differentiation for Selfie-dPCR
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To test the ability of Selfie-dPCR to differentiate between RNA and DNA, we treated samples with RNase A or with double-strand-specific DNase. The amount of enzyme and the incubation conditions were carefully optimized to achieve maximum selectivity for the RNA and DNA, respectively, because none of the enzymes exhibited absolute selectivity for their target after incubation for a prolonged time. For RNase treatment, we added 0.04 µl of 10 mg/ml RNase A (DNase and protease-free, EN0531, Thermo-Fisher Scientific), and 2.46 µl of water to 0.5 µl of tissue lysate. For DNase treatment, we added 0.08 µl of dsDNase (double-strand-specific DNase, EN0771, Thermo-Fisher Scientific) and 1.92 µl of water to 0.5 µl of tissue lysate. The reactions were incubated for 2 min at 37 °C and 3 min at 90 °C, and the entire volume was used for the sample-primer pre-annealing step described in the Selfie-dPCR procedure.
3
EV Lysis and dsDNA Digestion
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In cell experiments, EVs fractions isolated from the culture medium of damaged CT26 cells were lysed by sonication using the ultrasonic cell disruptor (JY92-IIN, Guansen Biotechnology (Shanghai) Co., Ltd.). The process was repeated 6–8 times by 5 s pulse /5 s pause in an ice bath. To digest dsDNA inside EVs, dsDNase (EN0771, Thermo Scientific) was added to EVs fractions and incubated at 37°C for 30 min after sonication.
4
Immunofluorescence Staining Protocol
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Cell fixation and antibody staining were performed as previously described7 (link). Briefly, cells were fixed with ice-cold (−30 C) methanol for 15 minutes – when staining for centromeres, centrosomes, cGAS, Vimentin, β-actin, IRF3, or a-tubulin – or 4% paraformaldehyde – when staining for RelB, p65, STING, ssDNA, dsDNA, CoxIV, or β-catenin. Subsequently, cells were permeabilized using 1% triton for 4 minutes. See supplementary Table 1 for antibody information. For selective plasma membrane permeabilization used for cytosolic dsDNA and ssDNA staining, cells were treated with 0.02% saponin for 5 minutes after fixation. For single-stranded (Thermo Fisher FEREN0321) and double stranded (Life Technologies – EN0771)-specific nuclease treatment, cells were also permeabilized with 0.02% saponin for 2 minutes and treated with either nucleases for 10 minutes before fixation using 4% paraformaldehyde. TBS-BSA was used as a blocking agent during antibody staining. DAPI was added together with secondary antibodies. Cells were mounted with Prolong Diamond Antifade Mountant (Life Technologies – P36961). cGAMP (Invivogen – tlrl-nacga23) was transfected into cells at a concentration of 4μg/ml using lipofectamine2000 that was added for 3–4 hours and then replaced with regular serum containing medium.
5
Immunofluorescence Staining Protocol
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