Ls 6000ic

To measure proliferation, B cells were incubated for 3 days and pulsed with 0.5 μCi of tritiated thymidine ([³H]-TdR; MP Biomedical, Irvine, CA) for the last 18 hours of culture. Cells were harvested onto fiberglass filter mats (Skatron Instruments, Sterling, VA) with a Titertek Cell Harvester (Skatron Instruments). [³H]-TdR uptake was assessed using a Beckman LS6000IC liquid scintillation counter (Beckman Coulter Inc., Mississauga, ON) and quantified as counts per minute (CPM). The values of each experiment were taken from the average of triplicate wells.

Samples for determination of prokaryote abundance (10 mL) by flow cytometry were fixed with formaldehyde (final concentration 3.7%) and stored at 4°C. Prokaryote activity was assessed by the micro-centrifuge method (Kirchman, 2001 (link)) with some modifications. Briefly, ³H-leucine (40 μM final conc.) was added to three replicated samples (10 mL) from each single depth. Additionally, one formaldehyde-killed blank for each depth was fixed at the sampling site. ³H-leucine incubations were done in the dark at the in situ water temperature (ca. 10°C) for 30 min and they were terminated by adding formaldehyde. Samples were filtered onto white 0.22-μm polycarbonate filters (Poretics). The samples were extracted with cold trichloroacetic acid (5%) for 5 min and rinsed 3 times with the same solution. Filters were placed in scintillation vials with 5 mL of scintillation cocktail (Ready-safe, Beckman Coulter, Brea, CA, United States). The radioactivity of the filters was assessed on a scintillation counter (LS 6000IC, Beckman Coulter).

Fresh kidney homogenates (~5 mg) were incubated in 3 mL of reverse Krebs–Henseleit bicarbonate medium with or without rotenone and antimycin A (10 + 50 μmol/L), blockers of mitochondrial respiratory system. Mitochondrial and peroxisomal fatty acid oxidations were measured in the medium using either [1-¹⁴C]-labeled oleic acid (C18:1) or erucic acid (C22:1) purchased from American Radiolabeled Chemicals (ARC; Saint Louis, MO, USA) as substrate. The biochemical and radio-chemical purities of both C18:1 and C22:1 were greater than 99% based on TLC and HPLC analyses. The fatty acids were bound to fatty acid-free BSA (5:1, molar ratio) and dissolved in the reaction medium. The measurements were performed in 25 mL Erlenmeyer flasks containing 2 mL of the reaction medium. The medium was incubated with 2 µmol [1-¹⁴C]-C18:1 (0.98 kBq/µmol) or [1-¹⁴C]-C22:1 (1.37 kBq/µmol). The incubation was stopped after 30 min by the addition of 0.5 mL of 35% HClO₄. The ¹⁴C accumulation in CO₂ and acid-soluble products (ASP) were collected, processed, and analyzed by liquid scintillation spectrometry (Beckman LS 6000IC, Fullerton, CA, USA) according to the procedures by Lin et al. [24 (link)].

Cultures performed in 96-well U-bottom plates were pulsed with 0.25 μCi of methyl ³H-thymidine ([³H]TdR; MP Biomedicals, Irvine, CA, United States) for 6 h. DNA was then harvested onto glass fiber filter mats using a Titer-Tek cell harvester (Skatron Instruments, Lier, Norway). [³H]TdR incorporation, which is a measure of DNA synthesis, was determined using a Beckman LS6000IC liquid scintillation counter (Beckman Coulter, Inc., Brea, CA, United States).