Quant it oligreen ssdna kit

Bioactive factors like IL2, GMCSF, FLT3L, and CpG were incorporated into cryogels by adding them to the gel mixture before cryogelation. To achieve controlled release, recombinant mouse IL2 (Biolegend #575408) and recombinant murine GM-CSF (Peprotech #315-03) or recombinant murine FLT3L (Peprotech #250-31L) were preloaded onto charged laponite XLG (BYK additives) as follows. First, laponite was dissolved at 30 mg/ml in milliQ with extensive vortexing until the solution became clear. For cryogels loaded with both IL2 and GM-CSF/FLT3L, 5 µg of IL2/50 µl gel solution and 1 µg of GM-CSF or FLT3L/50 µl gel solution were each separately adsorbed onto 25 µg laponite for 1 h at 4 °C. For cryogels loaded with IL2 only, 5 µg of IL2/50 µl gel solution was adsorbed onto 50 µg laponite for 1 h at 4 °C. This kept the total amount of laponite in the scaffold at 50 µg. 50 µg of CpG ODN 1826 (Invivogen #tlrl-1826-1) was adsorbed onto 3 µg linear 25 K PEI (Polysciences 23966) for 1 h at 4 °C. The preloaded bioactive factors were then added to the gel mix before the gels underwent cryogelation as described above.
CpG release studies were performed using the Quant-iT OliGreen ssDNA Kit (Thermo #O11492), while cytokine release was quantified via ELISA.

CpG modified with an amine group on 3′-end (0.75 nmol/µL) was reacted with 4FB (162 nmol/µL in DMSO) at a molar ratio of 1:20 in the modification buffer (0.1 M NaPO4, 0.15 M NaCl, pH 8.0) for 4 h at room temperature. After the reaction was complete, the unreacted linker was removed from 4FB-modified CpG by five cycles of centrifugal filtration using a spin filter (Vivaspin 500, MWCO 3 kDa, Satorius) and was resuspended in reaction buffer (0.1 M NaPO4, 0.15 M NaCl, pH 6.0).
aDNP (4 mg/mL, 400 µg aDNP dry mass) was reacted with a 40-fold molar excess of the HyNic linker (68.9 nmol/µL in DMSO) in the modification buffer for 3 h at room temperature. The unreacted linker was removed from HyNic-modified aDNP by five cycles of centrifugal filtration using a spin filter (Vivaspin 500, MWCO 3 kDa, Satorius) and was resuspended into the reaction buffer.
The HyNic-modified aDNP was reacted with 4FB-modified CpG at a molar ratio of 1:5 for 3.5 h at room temperature. The CpG-conjugated aDNP was washed in PBS buffer (pH 7.4) by 5 cycles of centrifugal filtration (Vivaspin 500, MWCO 10 kDa). The concentration of conjugated CpG was quantified using the Quant-iT OliGreen^® ssDNA kit (Thermo Fisher Scientific).

Per each enrolled patient, peripheral blood samples were collected in 2 × 4.9 mL clot activator tubes (S-Monovette Sarstedt) before the start of SBRT, and kept at room temperature for 30–60 min to allow for clotting, and then centrifuged at 2000× g for 15 at 4 °C. Circulating cell free DNA (cfDNA) was purified from serum using QIAamp Circulating Nucleic Acid kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cfDNA was quantified using QuantiT™ Oligreen^® ssDNA kit (Thermofisher, Waltham, MA, USA) and Infinite200 plate reader (Tecan, Männedorf, Switzerland).

aDNP (0.8 mg/mL based on dry mass, 1 mL) suspended in deionized water was mixed with either 4, 8, 16, 32, or 48 µL of CpG at a concentration of 5 µg/µL in deionized water followed by a gentle shaking (100 rpm) at room temperature for 4 h. The unbound CpG was then washed with PBS buffer (pH 7.4) by five cycles of centrifugal filtration (Vivaspin 500, MWCO 10 kDa, Sartorius, Göttingen, Germany) at 12,000× g, 4 °C. The washed nanoparticle suspension was then quantified for CpG using the Quant-iT OliGreen^® ssDNA kit (Thermo Fisher Scientific).

Total RNA was isolated from frozen whole quadriceps muscles using the TRIzol® protocol as previously described [9 (link)]. Concentrations and purity of RNA were determined by UV absorbance at 260/280 nm using a Nanodrop® 1000 spectrophotometer. Integrity of RNA was determined by running 1 μg of isolated RNA on an agarose gel with visualisation under UV light (GelDoc). Total RNA (2 μg) from each sample was reverse transcribed (RT) using oligo (dt) primers and SuperScript® III reverse transcriptase (Life Technologies, Carlsbad, California, USA) as per the manufacturer’s instructions. RT reactions were diluted 10-fold, and real-time PCR was performed using a Roche LightCycler® 2.0 as previously described [9 (link)]. The sequences of primers used and size of the amplicons are listed in Additional file 2: Table S2. Concentrations of target cDNA were normalised to concentrations of total ssDNA for each RT sample using a Quant-iT™ Oligreen® ssDNA kit (Life Technologies) as per the manufacturer’s instructions [27 (link)].