Carbonic anhydrase activity assay kit

Organoids were seeded in 6 well plate. At the time of analysis, culture media were aspirated then 1ml ice-cold PBS was added into one well. Pipette several times to physically disrupt the Matrigel and transfer the slurry into 15ml conical tube. Centrifuge tubes at 3500 rpm x 5minutes. Aspirate the supernatant carefully to not disrupt Matrigel layers. Add 1ml of ice-cold Cell Recovery Solution (Corning) and pipette several times then incubated tubes on ice for 1 hour. After the incubation, centrifuge tubes at 3500rpm x 5 minutes and aspirate the supernatant carefully not to disrupt cell pellets. Add 1ml of PBS to wash the pellet then centrifuge tubes at 3500rpm x 5 minutes and aspirate the supernatant carefully not to disrupt cell pellets. Pellets were lysed with enzyme analysis buffer (150 mM NaCl, 10 mM Tris, 1% Triton X-100, pH = 7.2) with no protease inhibitors supplemented. The protein concentration of cell lysates was determined by Bradford assay. Enzyme activities were analyzed using Lipase Activity Fluorometric Assay Kit (#K724, Biovision) and Carbonic Anhydrase Activity Assay Kit (#K472, Biovision), following the instructions by the manufacturer.

HPDE6c7 cells (RRID:CVCL_0P38) were authenticated by ATCC and tested negative for mycoplasma using a kit from InvivoGen (rep-pt1). HPDE6c7 cells (Furukawa et al., 1996 (link)) were cultured in DMEM (Life Technologies 11995073), 10% FBS (Corning, 35011CV), 1X Penicillin:Streptomycin solution (Corning, 30-002-CI). For cell counting, 25,000 cells were seeded in a sterile six-well TC-treated plate (Corning, 353046). Values depicted for all cell culture experiments represent the average of at least three independent experiments.
For carbonic anhydrase activity assays, cell lysates were prepared using standard protocols and cell lysis buffer (Cell Signaling Technologies, 9803S) containing 100 mM PMSF, 1X cOmplete Protease Inhibitor Cocktail (Roche, 11697498001), and 1X PhosSTOP (Sigma Aldrich, 4906845001). Carbonic anhydrase activity was measured using the carbonic anhydrase activity assay kit (Biovision, K472-100). For normalization, equal amounts of protein (10 µg) per sample were used in the assay. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225).