Elisa kit

Serum separator blood tubes were centrifuged at 1800 × g for 15 min at 20 °C (VACUETTE, Greiner Bio-One, UK); lithium heparin and ethylenediaminetetraacetic acid (EDTA) blood collection tubes (VACUETTE, Greiner Bio-One, UK) were centrifuged at 1800 × g for 10 min at 4 °C. Samples were kept at −20 °C for further analyses. TAG, glucose and non esterified fatty acids (NEFA) were quantified in serum by using an autoanalyser (ILAB600, Werfen (UK) Ltd, Warrington, UK; reagents and analyser: Instrumentation Laboratory Ltd.; NEFA reagent: Alpha Laboratories). An ELISA kit was used to determine serum insulin (Dako Ltd.). Serum angiotensin-converting enzyme (ACE) activity was determined by a fluorometric assay as described elsewhere^{25 (link)}.

Urine samples collected over 24 h in metabolic cages were centrifuged at 3000–6000g, and the supernatant was used to measure urinary protein, creatinine, and other parameters. Blood samples were collected under isoflurane anesthesia from the retroorbital venousplexus (at weeks 0, 1, and 2) using heparin-coated glass capillaries (Terumo Corporation, Tokyo, Japan) or the abdominal vena cava (at week 3) with 19- to 23-gauge needles and centrifuged at approximately 15,000g to measure the levels of serum or plasma creatinine, CCL2, and other substances in the supernatant. Heparin was used as the anti-coagulation reagent.
The kidneys were weighed after extraction from the body; in one of the kidneys, the upper half of the renal tissue was immersed in 10% neutral-buffered formalin for histological evaluation, and the cortex of the remaining half was frozen in liquid nitrogen and stored at − 80 °C until processing for mRNA quantification. The other kidney was lysed with Pierce™ IP Lysis Buffer (Thermo Fischer Scientific) containing 1× of Halt™ Protease Inhibitor Cocktail, EDTA-free (Thermo Fischer Scientific) to measure kidney CCL2 using an ELISA Kit (DAKO, Inc.) and kidney IFN-γ using a rat IFN-gamma Quantikine ELISA Kit (R&D systems, Minneapolis, MN, USA).

The serum and urine samples were collected soon after admission to the hospital. The collected samples were centrifuged at 3,000 g for 10 min to remove cellular debris. We aliquoted serum and urine supernatant into bar-coded cry-vials and stored the samples at −80°C until biomarker measurement. No additives or protease inhibitors were added.
We measured the serum and urine biomarker C4d with a commercially available ELISA kit (Dako, Denmark) following manufacturer's instruction. We measured urine creatinine by the modified Jaffe reaction. Personnel performing the biomarker measurements were blinded to each patient's clinical information. All biomarkers were measured from frozen aliquots that did not undergo any additional freeze-thaw cycles.

The differentiated cells at stage 3 were rinsed twice in Krebs-Ringer bicarbonate HEPES (KRBH) buffer. The cells were then incubated in KRBH buffer containing 5, 20, and 50 mM glucose at 37°C for 1 h, respectively. Supernatant were collected for insulin secretion measurement. Insulin levels were determined by insulin enzyme-linked immunosorbent assay (ELISA) kit (Dako). The hES-IPCs population that can secrete insulin in a glucose dependent manner were separate into 2 parts. One part subjected for alginate encapsulation and another part remain non-encapsulation.

The encapsulated hES-DIPCs were cultured at 37°C, 5% O₂, 4.5% CO₂ and 95% humidity. Media samples were collected at day 0, 2, 4, 6, 8, 10, 12, and 14 to assess the rate of insulin secreted by the encapsulated cultures. Insulin levels were determined by insulin enzyme-linked immunosorbent assay (ELISA) kit (Dako).

Endothelial function was assessed by flow mediated dilation (FMD) measurement as described and validated by our laboratory before [17] (link), [18] (link). Measurements were performed with the Toshiba Xario Diagnostic Ultrasound System and an 8MHz linear transducer. Briefly, measurements were recorded before 5 minute-long brachial artery occlusion and then subsequently 1, 2 and 5 minutes after cuff deflation. Measurements during diastole were recorded and maximal dilation (usually detected within ca. 60–120 sec) was analyzed and reported. Maximal nitroglycerine mediated dilation (NMD) was measured to study non-endothelium dependent vasodilations. vWF concentration in plasma was analyzed using a commercial ELISA kit from DAKO (Glostrup, Denmark).