Human mouse total cox 2 duoset ic elisa

Splenocytes from DBA/1 mice were incubated in the culture medium for 72 h in 24-well culture plates and a density of 5 × 10⁶/mL. The cells were stimulated with LPS (20 µg/mL) and RM33 was used at 10 µg/mL. The control cultures contained concentration of DMSO in the culture medium corresponding to the concentration of DMSO in cultures containing RM33.
After the incubation, the cells were detached by treatment with 0.1% trypsin solution for 10 min followed by 3× wash in PBS. The dry cell pellets were suspended in 0.5 mL of lysis buffer consisting of 1 mM EDTA, 0.5% Triton X-100 and containing 10 mg/mL leupeptin, 10 mg/mL pepstatin, and 3 mg/mL aprotinin in PBS, pH 7.4. Lysis was carried out on the ice for 15 min. Lysates were preserved by being deep-frozen (−80 °C). Before use, the lysate samples were centrifuged at 2000× g for 5 min, and the supernatant was transferred to a new tube. Sample total protein concentration was determined by the spectrophotometric method for equilibrating lysates dilutions. The content of cyclooxygenases in cells was determined by ELISA kits (Human/Mouse Total COX-2 DuoSet IC ELISA, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Briefly, HUVECs were cultured in a 6-well plate at a density of 5 × 10⁶ cells mL⁻¹ and pretreated with peptide fractions (20 µg mL⁻¹) for 24 h and then treated with 1 µg mL⁻¹ LPS for 12 h. Later, the culture medium was collected and stored at ≤ −70 °C. The cells were rinsed two times with PBS and then lysed within the wells by the addition of the Cell Extraction Buffer. After centrifugation at 2000× g for 5 min, cell lysates were stored at ≤ −70 °C. IL-1β released in the media and COX-2 cellular levels were determined using specific ELISA kits (cat. E00021, Human IL1-beta ELISA Kit, Proteintech Group, Inc, Rosemont, IL, USA; Catalog #: DYC4198-2, Human/Mouse Total COX-2 DuoSet IC ELISA, R&D Systems, Inc., Minneapolis, MN, USA), as per the manufacturer’s directions. Detection was carried out using Gen5 software (BioTek Instruments, Inc., Winooski, VT, USA).