Millex ha filter

Cores were sectioned for pore-water collection in an anaerobic glove box (N₂ atmosphere with 3–5% H₂; Coy lab products, USA). Cores were sectioned at 0.5 cm resolution from 0 to 6 cm depth and 1 cm resolution between 6 and 12 cm depth. Sediment slices were collected in 50 ml polypropylene centrifuge tubes (TPP, Switzerland) and centrifuged at 3,000 rpm for 10 min (Sigma 3–18KS, Sigma Laborzentrifugen GmbH, Germany). Subsequently, the centrifuge tubes were opened in the anaerobic glove box, and overlying pore water was transferred into suitable sample containers after filtration through 0.45 μm cellulose filters (Millex-HA filter, Merck Millipore, USA). Pore-water samples were analyzed for dissolved iron and dissolved As. To prevent oxidation, the solid phase that remained after centrifugation was freeze-dried and sealed in an airtight aluminum bag inside the anaerobic glove box and stored anaerobically for later solid-phase analysis.

HEK293T cells (ATCC CRL-3216) were seeded on 10 cm² dishes and allowed to adhere for 36 hours. The cells were transfected with 4 μg of pMD2.G (Addgene #12259), 4 μg of psPAX2 (Addgene #12260) and 8 μg of purified pLKO.1 plasmid containing the shRNA constructs upon reaching 70% confluency. Cell supernatant containing lentivirus was harvested 72 h post-transfection, filtered through a 45 μM Millex HA-filter (Merck Millipore) and purified by ultracentrifugation at 27,000x g for 90min. 2x10⁵ Calu-3 cells were seeded onto collagen coated 6-well plates 24 h prior to transduction. Cell medium was replaced with 3 mL medium containing 20 μL of the purified lentivirus and 3μl polybrene transfection reagent (Merck Millipore). Medium was supplemented with 10 μg/mL puromycin for selection of successfully transduced cells two to three days after transduction.

For water content determination, fresh soil samples were weighed and subsequently
dried at 105 °C until weight stability. pH was measured
in a suspension of 10 g air dried soil in 25 mL of a
0.01 M CaCl₂-solution. For determination of leachable
chloride and leachable organic carbon, 10 g of soil were mixed with
100 mL deionized water and shaken at 150 rpm for
24 h on a rotary shaker. Samples were centrifuged for
5 minutes at 4000 × g and
filtered through a 0.45 μm pore size cellulose ester
filter (Millex HA filter, EMD Millipore Corporation, USA). Dissolved organic
carbon was measured with a High TOC Elementar system (Elementar Analysensysteme
GmbH, Hanau, Germany) and chloride was determined by ion chromatography (Dionex
DX 120, Thermo Scientific, Sunnyvale, CA, USA). For total organic carbon
analysis soil samples were dried at 40 °C and sieved
(2 mm mesh) to exclude large roots and stones. The organic carbon
content was determined by heat combustion (1150 °C) and
thermal conductivity analysis on a CNS element analyzer (Elementar Vario EL III,
Elementar Analysensysteme GmbH, Hanau, Germany). Adsorbable organic halogen
(AOX) content in the soil samples was determined according to the standard
protocol (DIN EN ISO 9562) for soil leachates (DIN EN 12457-4) at the Laboratory
for Environmental and Product Analytics (DEKRA GmbH, Halle, Germany).

According to Pelusi et al. [32 (link)], 50 mL of each cell culture sample was harvested during the exponential growth phase. Half of the culture sample (25 mL) was centrifuged (1720× g, 5 min) to remove cells. Then, the supernatant was filtered using a Millex HA filter (pore size 0.45 μm, Merck Millipore, Darmstadt, Germany) to acquire culture medium without any particles, including cell debris (culture medium fraction). Total organic carbon (TOC) and total inorganic carbon (TIC) values of both the cell suspension and culture medium fraction (25 mL each) were measured using a TOC analyzer (Shimadzu Corporation, Kyoto, Japan). TIC was calculated by subtracting TOC from the total carbon. The cellular organic and inorganic carbon quantities (Cell C and Cell IC, respectively) were calculated using the following equations:

where the TOC and TIC values are expressed as cells contained in a 1-L culture (mg/L).

The yeasts were individually grown on banana agar three times. 100 mg of cells were scraped from the surface of the plates and snap-frozen. To prepare yeast-conditioned plates, yeasts were grown as described and thoroughly removed by scraping. 22 mm² chunks of agar from the plates (~100–160 mg) were collected and snap-frozen. 15 g/l agar plates or sterile banana-agar plates were prepared as controls and collected using the same procedure. To prepare cell suspension supernatants,~250 mg of yeast cells were collected as described, and sterile PBS was added at a ratio of 5 µl PBS per 1 mg of yeast cells. After suspending the cells, the suspensions were centrifuged at 2260 × g for 5 min. The supernatants were further filtered using a 0.45-µm Millex-HA filter (Merck) to completely remove any remaining yeast cells. 700 µl of each supernatant was collected and snap-frozen.
Each sample was suspended or diluted to 500µl of methanol containing internal standard; 30 μM 2-Morpholinoethanesulfonic acid and 30 μM L-Methionine. After mixing with 250 µl of water and 400 µl of chloroform, the samples were centrifuged and the upper layer was collected and filtered using an UltrafreeMC-PLHCC for Metabolome Analysis column (Human Metabolome Technologies, #UFC3LCCNB-HMT). The samples were dried completely using nitrogen gas and resuspended in water before injection into the LC–MS system.