Human c peptide elisa kit

Cells at D30_L were dissociated using Accutase and resuspended in DMEM containing 2% FBS and 1 mM EDTA; 20,000 INS-GFP⁺ DAPI⁻ cells were FACS sorted by an ARIA2 instrument, washed once with PBS and lysed in 200 µL RIPA buffer supplemented with 1× protease inhibitor cocktail (ThermoFisher Scientific). The Insulin content in the lysates was measured by ELISA (Human C-peptide ELISA kit, Millipore, EZHCP-20K).

To analyze the levels of C-peptide released from intravitreal K9-C-peptide depots, mice were anesthetized with 3% isoflurane and intravitreally injected with a total volume of 2 μL containing 0.6 mg/mL (1.2 μg) C-peptide or 10 mg/mL (20 μg) K9-C-peptide. Proteins were extracted from whole eyeballs using lysis buffer containing 50 mmol/L Tris (pH 7.5), 150 mmol/L sodium chloride, 1% Triton X-100, protease inhibitor cocktail (Roche, Basel, Switzerland), 25 mmol/L β-glycerophosphate, and 2 mmol/L sodium orthovanadate. The lysates were centrifuged at 14,000 rpm for 15 min, and then the supernatants were centrifuged at 8,000 rpm for 10 min using Amicon^® Ultra 4 mL centrifugal filters with a molecular weight cutoff of 50 kDa (MilliporeSigma). C-peptide levels in the filtered supernatants (n = 6) were measured using a human C-peptide ELISA kit (MilliporeSigma) according to the manufacturer's protocol.

Insulin and C-peptide levels in mouse serum were measured using a human insulin ELISA kit (MilliporeSigma, Burlington, MA, USA), a mouse C-peptide ELISA kit (Alpco, Salem, NH), and a human C-peptide ELISA kit (MilliporeSigma), respectively, according to the manufacturer’s instructions as previously described [26 (link)]. The blood levels (n = 6) of insulin and C-peptide were determined by measuring absorbance at 450 nm using a microplate spectrophotometer (Epoch; BioTek, Winooski, VT, USA).

For intravitreal fluorescence imaging, K9-C-peptide and human C-peptide were conjugated with NHS-Fluorescein (Thermo Fisher Scientific, Waltham, MA, USA), as previously reported 21 (link). Briefly, K9-C-peptide (100 μL, 50 mg/mL) or C-peptide (100 μL, 1 mg/mL) in 100 mmol/L sodium bicarbonate buffer (pH 8.3) was mixed with 45 μl or 16 μL, respectively, of 10 mg/ml NHS-Fluorescein in 10% dimethyl sulfoxide and incubated for 2 h on ice. To quench the reaction, 7 μL of 1 mol/L Tris-HCl (pH 8.0) was added to the reaction solution. The reaction mixtures were loaded onto 1.5 mL Sephadex G-25 columns, and fluorescein-conjugated K9-C-peptide and C-peptide were eluted by centrifugation for 3 min at 1,050 × g. The levels of conjugated K9-C-peptide and C-peptide levels in the elutes were determined using a human C-peptide ELISA kit (MilliporeSigma).
To analyze the distribution of the polypeptides in vivo, mice were anesthetized with 3% isoflurane and intravitreally injected with a total volume of 2 μL containing 0.6 mg/mL (1.2 μg) fluorescein-conjugated C-peptide or 5.0 mg/ml (10 μg), 7.5 mg/mL (15 μg), 10.0 mg/mL (20 μg), or 12.5 mg/mL (25 μg) fluorescein-conjugated K9-C-peptide. Intravitreal fluorescence imaging was performed using confocal microscopy (K1-fluo; Nanoscope Systems, Daejon, Korea). Polypeptide levels were quantitatively analyzed by measuring fluorescence (n = 6).