Human islets from a subset of donors (n = 45) were cultured overnight in islet culture medium, trypsinized to single cells using and plated on uncoated glass coverslips (Fisherbrand) as previously described in Kong et al. 201812 (link). Dispersed cells were cultured in islet culture medium containing either 5 or 15 mmol/L glucose and 20 μg/mL bromodeoxyuridine (Sigma) for 96 h. After culture, the islet cells were fixed for 10 min in 4% paraformaldehyde (Sigma). Fixed cells were unmasked in 1 N HCl for 20 min at 37 °C, blocked for 2 h in goat serum–based block with 0.1% Tween 20. Immunofluorescence staining was performed for insulin (ab7842, Abcam, or A056401-2; Dako), and BrdU (ab6326; Abcam) antibodies, and DAPI (Sigma) as previously described12. The percent of insulin-staining cells that were also BrdU labeled, was quantified on blinded images to calculate β–cell proliferation. Representative images of data are shown in Fig. S9 of Kong et al. 201812 (link). Data were expressed as the proliferation index, calculated using the following ratio.
Uncoated glass coverslips
Manufactured by Thermo Fisher Scientific
Uncoated glass coverslips are thin, transparent glass plates used to cover and protect samples in microscopy applications. They provide a clear surface for mounting and observing specimens under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using uncoated glass coverslips
1
Quantifying Human Islet Cell Proliferation
2
Proliferation Assay of Human Islet Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proliferation assays. Human islets from a subset of donors (n=45) were cultured overnight in islet culture medium, trypsinized to single cells using and plated on uncoated glass coverslips (Fisherbrand) as previously described (12) . Dispersed cells were cultured in islet culture medium containing either 5 mmol/L or 15 mmol/L glucose and 20 μg/mL bromodeoxyuridine (Sigma) for 96 hours. After culture, the islet cells were fixed for 10 min in 4% paraformaldehyde (Sigma). Fixed cells were unmasked in 1N HCl for 20 min at 37°C, blocked for 2 hours in goat serum-based block with 0.1% Tween 20.
Immunofluorescence staining was performed for insulin (ab7842, Abcam, or A056401-2; Dako), and BrdU (ab6326; Abcam) antibodies, and DAPI (Sigma) as previously described (12) . The percent of insulin-staining cells that were also BrdU labeled, was quantified on blinded images to calculate β-cell proliferation. Data were expressed as the proliferation index, calculated as the ratio of %BrdU+ β-cells in 15 mmol/L glucose divided by the %BrdU+ β-cells in 5 mmol/L glucose. assays (Thermo Fisher Scientific, Waltham, MA) as previously described (12) .
Immunofluorescence staining was performed for insulin (ab7842, Abcam, or A056401-2; Dako), and BrdU (ab6326; Abcam) antibodies, and DAPI (Sigma) as previously described (12) . The percent of insulin-staining cells that were also BrdU labeled, was quantified on blinded images to calculate β-cell proliferation. Data were expressed as the proliferation index, calculated as the ratio of %BrdU+ β-cells in 15 mmol/L glucose divided by the %BrdU+ β-cells in 5 mmol/L glucose. assays (Thermo Fisher Scientific, Waltham, MA) as previously described (12) .
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!