10 kda filter

NGO was prepared according to the modified-Hummer’s method, starting with the oxidization of graphite sheets, followed by ultrasonication [37 (link)]. NGO-PEG (PG) and the NGO-PEG-BPEI (PPG) were prepared according to our previously described method [28 (link)–31 (link)]. To conjugate TPP with PPG, 5 mg of TPP was dissolved in 4 mL water and activated using EDC·HCl (15 mg) and NHS (15 mg) for 15 min at room temperature. Subsequently, 4 mL of PPG (1.0 mg/mL) solution was added to the reaction mixture and magnetically stirred at room temperature for 24 h. Finally, excess TPP was removed via filtration through a 10-kDa filter (Millipore, Inc.) and washed repeatedly with double-distilled water to obtain PPG-TPP (NGO-PEG equivalent, 0.5 mg/mL).
To synthesize TPP-PPG@ICG, ICG (7.74 mg) was dissolved in 1 mL of anhydrous dimethyl sulfoxide as a stock solution (10 mM) for further use. Two hundred microliters of ICG (10 mM) and 1.8 mL of TPP-PPG (0.5 mg/mL) were mixed and stirred for 24 h at room temperature. Then, the whole system was dialyzed against distilled water for 24 h (molecular weight cut-off: 10 kDa). The final product (TPP-PPG@ICG) was freeze-dried and stored below 4 °C for further use.

Antibody labeling with the indicated metal tag was performed using the MaxPAR antibody conjugation kit (Fluidigm) according to the manufacturer’s instructions. Purification of the bound antibody was performed with high-performance liquid chromatography (Thermo Fisher) and subsequently concentrated by filtering with a 10-kDa filter (Merck Millipore) in a swing-out bucket at 4000 RPM for 15 min. The end volume was determined and an equal volume of antibody stabilizer buffer (Fluidigm; supplemented with 0.05% sodium azide) was added before the antibodies were stored at 4 °C. All antibodies used in this study were titrated using both fixed and unfixed thawed immune cells and the most optimal concentrations with the least spillover were chosen. We adjusted the concentration accordingly for our frozen antibody cocktail aliquots.

Extraction of enzymes from the compost was tested under various conditions such as pH, extraction time and temperature and, finally, a modified method from Singh et al. (2003) [10 ] was used in order to achieve the highest recovery of proteins from the compost.
Frozen compost samples were defrosted and on the same day, 10 g of the sample was mixed with 100 mL distilled water in 250 mL Erlenmeyer flasks. The flasks were incubated for 1 h at 200 rpm and 4°C. Samples were then centrifuged (10 000 x g, 15 min, 4°C) and the supernatant was collected as the crude enzyme extract, which was used for PNP assays (2.4). For the other assays, supernatants were filtered through 0.2 μm filters. The filtrate obtained was then filtered through a 10 kDa filter (Merck Millipore, Billerica, MA, USA) and washed twice to remove small carbohydrates. The 10 kDa retentate was mixed with millipore water to reach the starting volume and denoted as “enzyme extract" from corresponding compost phases.

Antibody labeling with the indicated metal tag was performed using the MaxPAR antibody conjugation kit (Fluidigm) according to the manufacturer’s instructions. Purification of the bound antibody was performed with high-performance liquid chromatography (Thermo Fisher) and subsequently concentrated by filtering with a 10 kDa filter (Merck Millipore) in a swing-out bucket at 4000 RPM for 15 min. The end volume was determined and an equal volume of antibody stabilizer buffer (Fluidigm; supplemented with 0.05% sodium azide) was added before the antibodies were stored at 4 °C. All antibodies used in this study were titrated using both fixed and unfixed thawed PBMCs and the most optimal concentrations with the least spillover were chosen. Concentrated antibody cocktails for surface and nuclear antigen detection were made, aliquoted, and stored at –80 °C, as previously described (Schulz et al., 2019 (link)).