3h choline chloride

To study the release of choline-containing PLs from FLS during treatment, confluent cells from Passage 5 were cultured in 6-well plates and labeled with 5 µCi/mL [³H]-choline chloride (PerkinElmer, Rodgau, Germany) for 24 h in choline-depleted, phenol red-free DMEM (PAN Biotech) that was supplemented as above. FLS were washed thoroughly to remove unincorporated precursors and starved for 24 h with supplemented DMEM that contained 5% FBS. After a change in media, cells were treated for 48 h with 0.1 µM T0901317 (Sigma-Aldrich, Taufkirchen, Germany) or GW3965 (Cayman Chemical Company, Ann Arbor, MI, USA); 1 ng/mL IL-1ß (PeproTech, Hamburg, Germany), and 1 µM of the P38 MAPK inhibitor SB203580, 0.1 µM of the NF-κB inhibitor QNZ, the broad-spectrum JNK inhibitor SP600125 (Selleck Chemicals, Munich, Germany), or a negative control. The media and treatment were changed after 24 h. The release of radiolabeled PLs was measured in media from the final 24 h of the experiment. The experiment was repeated 4–8 times using FLS from 5 to 9 OA patients (age 53–81 years (69.3 ± 8.2), BMI 26.8–34.9 kg/m² (30.1 ± 2.4), Kellgren-Lawrence scores of 2–4 (3.1 ± 0.8), CRP 3.18 ± 4.51 mg/L, 3 males, 6 females).

The rates of choline uptake, incorporation into PC, uptake inhibition and degradation were determined in M[0] and M[IL-4] using ³H-choline chloride (Perkin Elmer) as previously described [4 (link)].

The rate of choline uptake was determined by measuring 3H-choline chloride (Perkin Elmer) uptake over time. Platelets or mitochondria were added into 1.5 mL tubes. One hour prior to uptake, media were removed and cells were washed with PBS before being incubated in Tyrode’s buffer until use. Immediately prior to uptake, cells were washed again followed by the addition of Tyrode’s buffer containing 1 μCi/mL of 3H-choline and were incubated at 37 °C for desired time point (1–30 min). Following incubation, cells were washed twice with Tyrode’s buffer, lysed in 150 μL of 0.1 M NaOH and an aliquot was used to determine radioactivity by liquid scintillation counting. Total cellular protein was determined using a BCA protein assay (Thermo Fisher Scientific) according to the manufacturer’s instructions. Choline uptake was expressed as counts per minute per mg protein (for mitochondria) or per 50,000 cells (for platelets).