Hpg alexa fluor 488 protein synthesis assay kit

SW1353 cells were incubated in 6-well plates for 30 min with 25 µCi/mL EasyTag [35S] protein labeling mix (Perkin Elmer, Rotterdam, The Netherlands) in DMEM without methionine or cystine, and supplemented with 10% FCS and 1% P/S. Cells were carefully washed four times with 0.9% NaCl and lysed in 150 µL RIPA buffer. Lysates were sonicated in a water bath and centrifuged for 15 min at 13,200 rpm in an Eppendorf tabletop centrifuge. A quantity of 50 µL of cleared lysates was measured for 5 min on a scintillation counter (Tri-Carb 2910 TR, Perkin Elmer, Rotterdam, The Netherlands), and data were extracted as counts per minute beta. Data were normalized to total DNA content per well using a Sybr Green DNA assay as described earlier [73 (link)]. Alternatively, SW1353 cells were incubated in a 96-well plate for 24 h with 50 µM of the methionine analogue homopropargylglycine (HPG) in DMEM without methionine and cystine, and supplemented with 63 mg/L cystine (Sigma-Aldrich), 10% FCS and 1% P/S. HPG incorporation was detected with a Click-IT reaction (HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit, Thermo Fisher Scientific, Breda, The Netherlands) with a TriStar2 (Berthold Technologies, Vilvoorde, Belgium). Data were normalized to total DNA content per well using the included HCS Nuclear mask blue stain.

Protein synthesis in cultured chondrocytes was analyzed using a Click-iT L-Homopropargylglycine (HPG) Alexa Fluor 488 Protein Synthesis Assay Kit (Thermo Fisher Scientific) 72 h after cell seeding, as described previously.¹⁰ The mean fluorescence intensity of HPG-Alexa Fluor 488 was calculated using Flowing software.