After microglia removal, OPCs growing on top of astrocyte monolayer were isolated by shaking cells on an orbital shaker at 200 rpm for 3 h and incubated on an uncoated Petri dish for 1 h to further eliminate microglia. Pure OPCs (> 95% [32 (link)] were seeded onto poly-d ,l -ornithine-coated glass coverslips or plates (50 μg ml−1, Sigma-Aldrich, Milan, Italy) in Neurobasal (Life Technologies, Monza, Italy) supplemented with 2% B27 (Life Technologies, Monza, Italy), 2 mM l -glutamine (EuroClone, Milan, Italy), 10 ng ml−1 human platelet-derived growth factor BB (Sigma-Aldrich, Milan, Italy), and 10 ng ml−1 human basic fibroblast growth factor (Space Import Export, Milan, Italy), to promote proliferation (proliferating medium). After 3 days, cells were either detached with accutase (Millipore, Burlington, MA, USA) and used for migration assay, or switched to a Neurobasal medium lacking growth factors and supplemented with triiodothyronine T3 (10 ng ml−1, Sigma Aldrich, Milan, Italy) to allow differentiation (differentiating medium).
Human platelet derived growth factor bb
Manufactured by Merck Group
Sourced in Italy
Human platelet-derived growth factor BB is a recombinant protein that serves as a growth factor. It is a dimeric polypeptide composed of two identical B-chain subunits derived from human platelets.
Automatically generated - may contain errors
Lab products found in correlation
Human basic fibroblast growth factor, by Thermo Fisher Scientific (3 mentions)
Neurobasal, by Thermo Fisher Scientific (2 mentions)
Triiodothyronine t3, by Merck Group (2 mentions)
L glutamine, by Euroclone (2 mentions)
L glutamine, by Merck Group (2 mentions)
Accutase, by Merck Group (1 mentions)
Neurobasal, by Merck Group (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
5 protocols using human platelet derived growth factor bb
1
Isolation and Differentiation of Oligodendrocyte Progenitors
2
Culturing and Transfecting Rat OPCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mixed glial cultures were obtained from 12 postnatal day 2 (P2) Sprague-Dawley rat cerebral cortices pooled together as previously described (Lecca et al., 2016) (link). OPCs were plated onto poly-D,Lornithine-coated (final concentration 50 µg/ml; Sigma-Aldrich) 13-mm glass coverslips for immunocytochemistry (1-2 x 10 4 cells/coverslip) and poly-D,L-ornithine-coated 6-wells plates (10 5 cells/coverslip) or 6-cm dishes (2 x 10 5 cells/coverslip) for qRT-PCR assays. Cells were plated in Neurobasal medium supplemented with 2% B27 (Life Technologies), 2 mM L-glutamine, 10 ng/ml human platelet-derived growth factor BB (Sigma-Aldrich), and 10 ng/ml human basic fibroblast growth factor (Life Technologies) to promote proliferation. When OPCs reached a 60% confluency, cultures were switched to a Neurobasal medium lacking growth factors and containing triiodothyronine 15 nM (T3, Sigma-Aldrich) up to 4 days to allow differentiation. For transfection experiments, immediately after switching from proliferating to differentiating medium, OPCs were transfected with miR-125a-3p mimics (Dharmacon) or Gas7/Nod1 stealth siRNA (Life Technologies) at the final concentration of 50 nM with Lipofectamine RNAiMAX reagent (Life Technologies), following the manufacturer's protocol. A scrambled RNA transfection was included as negative control. Analysis were performed 48 hours after transfection.
3
Isolation and Culture of Primary Rat OPCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary rat OPCs were obtained from postnatal day 2 (P2) Sprague–Dawley rats’ cerebral cortices pooled together as previously described [27 (link),28 (link)]. OPCs were plated onto poly-D,L-ornithine-coated (final concentration 50 µg/mL; Sigma-Aldrich, Milan, Italy) 10 cm dishes (2–4 × 105 cells/coverslip) for metabolomic analysis, onto poly-D,L-ornithine-coated 13-mm glass coverslips for immunocytochemistry (1–2 × 104 cells/coverslip), and onto poly-D,L-ornithine-coated 6-well plates (7 × 104 cells/coverslip) for lipidomic and qRT-PCR analysis. Cells were plated in Neurobasal medium supplemented with 2% B27 (Life Technologies, Monza, Italy), 2 mM L-glutamine, 10 ng/mL human platelet-derived growth factor BB (Sigma-Aldrich, Milan, Italy), and 10 ng/mL human basic fibroblast growth factor (Life Technologies, Monza, Italy) to promote proliferation for 3 days.
4
Isolation and Culture of Rat Oligodendrocyte Progenitor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
OPCs were isolated from mixed glial cultures from postnatal day 2 Sprague-Dawley rat cortex, by the shaking method, as described [13 (link)]. OPCs were cultured in Neurobasal with 2% B27 (Invitrogen, Monza, Italy), 2 mM l -glutamine, 10 ng/mL human platelet-derived growth factor BB (Sigma-Aldrich, Milan, Italy), and 10 ng/mL human basic fibroblast growth factor (Invitrogen, Monza, Italy) to promote proliferation. About the 90% of cells was positive for the typical OPC marker Olig2; a very low percentage of contaminating astrocytes and microglia was found. After one day, cells were switched to a Neurobasal medium lacking growth factors to allow differentiation. Experiments were performed after Five to six days of differentiation in culture, when, in our standardized protocol [13 (link)], cells reached the immature pre-oligodendrocyte stage; indeed, previous studies [13 (link)] have shown that GPR17 expression is maximal in immature pre-oligodendrocytes and then gradually decreases along with terminal maturation.
5
Isolation and Culture of Primary OPCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary OPCs were isolated from mixed glial cultures prepared from postnatal day 2 Sprague-Dawley rat cortex by shaking cells on an orbital shaker at 200 rpm, as previously described (Fumagalli et al., 2011) . OPCs were then collected and separated from microglia by incubation for 1h on an uncoated Petri dish. Purified OPCs were seeded onto poly-D,Lornithine-coated glass coverlisps or plates (50 µg/ml, Sigma-Aldrich, Milan, Italy) to a specific density according to the planned experiments (see below), in Neurobasal (Life Technologies, Monza, Italy) with 2% B27 (Life Technologies), 2 mM L-glutamine (EuroClone), 10 ng/ml human platelet-derived growth factor BB (Sigma-Aldrich), and 10 ng/ml human basic fibroblast growth factor (Space Import Export, Milan, Italy), to promote proliferation. After 2 day, cells were switched to a Neurobasal medium lacking growth factors to allow differentiation. In some experiments triiodothyronine T3 (Sigma Aldrich)
was also added to a final concentration of 10 ng/ml.
was also added to a final concentration of 10 ng/ml.
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!