Rat islets were isolated by ductal Collagenase Perfusion and Histopaque gradient separation (Sigma). Pancreata were perfused with Hanks’ balanced salt solution supplemented with 1% BSA (wt/vol; Fisher), 4 mmol/L NaHCO3 (Sigma), and 10 mg/mL Collagenase P (Roche); excised; and incubated at 37°C for 15–30 min. After digestion, islets were washed and then purified using a Histopaque discontinuous gradient (Sigma). For fetal islet isolation, collagenase concentration was decreased to 2 mg/mL and incubated at 37°C for 10 min.
Histopaque gradient separation
Manufactured by Merck Group
Histopaque gradient separation is a laboratory product used to isolate and separate different types of cells, such as mononuclear cells, from whole blood samples. It is a solution that creates a density gradient, allowing the separation of cell types based on their specific densities.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit for rt pcr, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3 real time pcr instrument, by Thermo Fisher Scientific (2 mentions)
Qiaamp viral rna mini kit, by Qiagen (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Nahco3, by Merck Group (1 mentions)
Collagenase p, by Roche (1 mentions)
Maxima sybr green rox qpcr master mix, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Facsaria 3 flow cytometer, by BD (1 mentions)
4 protocols using histopaque gradient separation
1
Rat Islet Isolation Protocol
2
Plasma RNA Extraction and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood Serum was isolated from fresh blood samples collected from 3 healthy donors, 6 asymptomatic COVID-19 patients, 2 severe COVID-19 patients with pulmonary findings only, and 4 severe COVID-19 patients with pulmonary and extrapulmonary findings (i.e. elevated levels of creatinine and liver enzymes) following the approval of the ethical committee at by the Abu Dhabi Health COVID-19 Research Ethics Committee (DOH/DQD/2020/538), SEHA Research Ethics committee (SEHA-IRB-005) and Dubai Scientific Research Ethics Committee (DSREC-04/2020_09). The blood plasma was isolated using histopaque gradient separation (Sigma). Total RNA was extracted from 300µl of Plasma using QIAamp Viral RNA Mini Kit (Qiagen). cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit for RT PCR (Applied Biosystems). qRT-PCR was performed in triplicates with the Maxima SYBR Green/ROX qPCR Master Mix (Thermoscientific) using QuantStudio3 Real-Time PCR instrument (Applied biosystems). qRT-PCR were performed using primers for 18SrRNA, SOCS3, and TRIM56 as per the sequences in Table S2
3
Isolation and Co-culture of PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples collected from four healthy donors following the approval of the ethical committee at University Hospital Sharjah. The research ethics approval code for this study is UHS-HERC-033-02042020. The PBMCs were isolated using histopaque gradient separation (Sigma). PBMCs were labeled with 4 µM carboxyfluorescein succinimidyl ester (CFSE, Invitrogen, USA) for 8 min at room temperature and washed with ice cold RPMI 1640 medium (completed with 10% FBS and 1% penicillin-streptomycin). PBMCs were then directly co-cultured with a monolayer of A549 cells treated with 0, 5, and 10 µM of GN25 in complete RPMI 1640 medium at a seeding ratio of 1:1 (5×104 cells per well in a 12-well cell culture plate). PBMCs were harvested on the third day of culture and stained with anti-Human CD3-AlexaFluor 700 (clone OKT3, eBiosciences, Invitrogen, USA). Stained PBMCs were then acquired using BD FACS Aria III flow cytometer (BD Biosciences, USA) and BD FACS Diva software.
4
Plasma RNA Profiling in COVID-19 Patients
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood Serum was isolated from fresh blood samples collected from 4 healthy donors, 4 severe COVID-19 patients without liver dysfunction, and 9 severe COVID-19 patients with liver dysfunction (elevated alanine aminotransferase levels: > 41 U/L). The samples were collected following the approval of the ethical committee by Dubai Scientific Research Ethics Committee (DSREC-04/2020_09). Histopaque gradient separation (Sigma) was used to extract blood plasma and the RNA content was extracted from 300 µl of plasma using QIAamp Viral RNA Mini Kit (Qiagen). cDNA synthesis was carried out using the High-Capacity cDNA Reverse Transcription Kit for RT-PCR (applied Biosystems) and qRT-PCR was performed using about 50 ng of cDNA in triplicates with the Maxima SYBR Green/ROX qPCR Master Mix (Thermoscientific) using QuantStudio3 real-time PCR instrument (applied biosystems). qRT-PCR was performed using primers for 18SrRNA, DNAJB1, HSP90A1 and CYP39A1 as per the sequences in Table 2 .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!