Cspgs

Manufactured by Merck Group

Sourced in China, United States

CSPGs are a type of lab equipment used for the purification and separation of biomolecules. They utilize a chromatographic process to isolate and concentrate target analytes from complex mixtures. The core function of CSPGs is to facilitate the efficient and precise separation of biomolecules, such as proteins, nucleic acids, or other macromolecules, based on their unique physical and chemical properties.

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Lab products found in correlation

Close spelling variants of this product

CSPG mix Chondroitin sulfate proteoglycan (CSPG) Mouse anti-CSPG antibody

9 protocols using cspgs

Investigating Axonal Growth Inhibitors

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To explore the effect of specific inhibitors on axonal growth, 6-well plates were coated with 0.01% PLL overnight. The next day, they were washed three times with PBS and dried at 37°C. Then, 3-µl droplets of CSPGs (50 µg/ml; EMD Millipore) were spotted onto the 6-well plates for 4 h at 37°C. After the droplets dried, the six-well plates were covered with laminin(LN) (10 µg/ml; Invitrogen) for 2 h at 37°C. They were then washed three times with PBS and stored at 37°C before neuron plating.

Liu S., Jia J., Zhou H., Zhang C., Liu L., Liu J., Lu L., Li X., Kang Y., Lou Y., Cai Z., Ren Y., Kong X, & Feng S. (2019). PTEN modulates neurites outgrowth and neuron apoptosis involving the PI3K/Akt/mTOR signaling pathway. Molecular Medicine Reports, 20(5), 4059-4066.

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Dissection and Culture of Dorsal Root Ganglia

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DRGs from adult Thy1-ChR2 mice were dissected, washed in cooled Hank’s balanced salt solution (HBSS; ThermoFisher Scientific), and enzymatically digested (5 mg/ml Dispase II (Merck) and 2.5 mg/ml Collagenase Type II (ThermoFisher Scientific) in DMEM (ThermoFisher Scientific)) for 45 min at 37 °C. Next, the DRGs were resuspended in DMEM:F12 (ThermoFisher Scientific) media supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS; ThermoFisher Scientific) and 1× B27 (ThermoFisher Scientific) and were mechanically dissociated by pipetting. After centrifugation, the resulting single cells were resuspended in culture media (DMEM:F12 media with 1× B27 and penicillin/streptomycin (P/S; ThermoFisher Scientific)) and plated in glass-coverslips (3000–4000 cells/well) or in microfluidic devices (20.000 cells/device) pre-coated with 0.1 mg/ml poly-d-lysine (2 h, 37 °C; Merck) and 2 μg/ml laminin (over-night (O/N); ThermoFisher Scientific) at RT (room temperature)). An additional incubation with 5, 10 or 20 μg/ml CSPGs (Merck) was performed (2 h at 37 °C) in growth-inhibitory substrate experiments. Cells were allowed to grow for 24 h or for 8 days (in the case of microfluidic devices) at 37 °C in a 5% CO₂ atmosphere.

Mesquida-Veny F., Martínez-Torres S., Del Río J.A, & Hervera A. (2022). Genetic control of neuronal activity enhances axonal growth only on permissive substrates. Molecular Medicine, 28, 97.

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Neuronal Adhesion and Signaling Protocols

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Plates were precoated with PDL (20 μg/ml) overnight at room temperature (RT). In addition, some wells were coated with myelin or liver membranes at 10 μg/ml in PBS overnight at 4°C, or laminin (1 μg/ml; Sigma-Aldrich) for 4 hours at RT. In some experiments, 10 μM forskolin (Sigma-Aldrich), ERK-kinase-(MEK) inhibitor (10 μg/ml; PD98059), ERK inhibitor (12.5 μM; CAS1049738), or CSPGs (10 to 30 μg/ml; EMD Millipore) was applied to the culture medium. All cells were cultured at 37°C in 5% CO₂ for 48 hours, if not stated otherwise.

Poplawski G.H., Lie R., Hunt M., Kumamaru H., Kawaguchi R., Lu P., Schäfer M.K., Woodruff G., Robinson J., Canete P., Dulin J.N., Geoffroy C.G., Menzel L., Zheng B., Coppola G, & Tuszynski M.H. (2018). Adult rat myelin enhances axonal outgrowth from neural stem cells. Science translational medicine, 10(442), eaal2563.

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Optimizing Neuron Adhesion with Laminin and CSPG

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Coverslips were coated overnight in poly-L-ornithine (20 µg/ml; Sigma-Aldrich) and rinsed with sterile deionized H₂O the following day. The coverslips were subsequently coated overnight in three different concentrations (1, 5 and 10 µg/ml) of laminin (cat. no. L2020; Sigma-Aldrich; Merck KGaA), with CSPGs (cat. no. CC117; EMD Millipore, Billerica, MA, USA) at varying concentrations (1, 2.5, 5 and 10 µg/ml) or without CSPGs.

Sun Y., Deng Y., Xiao M., Hu L., Li Z, & Chen C. (2017). Chondroitin sulfate proteoglycans inhibit the migration and differentiation of oligodendrocyte precursor cells and its counteractive interaction with laminin. International Journal of Molecular Medicine, 40(6), 1657-1668.

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Immunohistochemical Analysis of Neural Markers

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5-Bromo-20-deoxyuridine (BrdU), glial fibrillary acidic protein (GFAP), ionized calcium binding adapter molecule 1 (Iba1), and neurofilament-200(NF200) antibodies were purchased from Abcam (MC, UK). Ras homolog gene family, member A (RhoA), anti-C3d, anti-NGR, interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor α (TNFα), growth associated protein-43 (GAP43), and anti-GAPDH antibodies were purchased from Affinity (OH, USA). S100 calcium-binding protein A10 (S100A10) and anti-ROCK2 were purchased from ABclonal Technology Co., Ltd. (Wuhan, China). CSPGs was purchased from Merck Chemicals (Shanghai, China) Co., Ltd. Neuron-specific nuclear protein (NeuN) was purchased from Novus (Co, USA). Antitubulin was purchased from Proteintech (IL, USA). Y-27632 dihydrochloride was purchased from APExBIO (Tx, USA).

Ying X., Yu X., Zhu J., Li X., Zheng Y., Xie Q., Wu Q., Li S., Yue J., Zhou Y., Zhou K., Tu W, & Jiang S. (2022). Water Treadmill Training Ameliorates Neurite Outgrowth Inhibition Associated with NGR/RhoA/ROCK by Inhibiting Astrocyte Activation following Spinal Cord Injury. Oxidative Medicine and Cellular Longevity, 2022, 1724362.

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Evaluating Axonal Growth of Cortical Neurons

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Primary cortical neurons were extracted from the embryos of pregnant Sprague-Dawley (SD) rats (E18) as previously described 30 (link). To evaluate the effects of DTX on axonal growth, primary cortical neurons were plated into poly-L-lysine-coated 12-well plates (Sigma Aldrich) with pre-equilibrated medium supplemented with 3.34 µg/mL CSPGs (chicken extracellular chondroitin sulfate proteoglycans, Millipore) dissolved in dimethyl sulfoxide (DMSO) at 1×10⁵ cells/well. After two days, the cells were treated with DMSO or varying concentrations of DTX (0.5 nM, 1 nM, 2 nM, 5 nM, 10 nM) for 72 h. To evaluate the effects of hydrogels, neurons were plated in 12-well plates (1×10⁵ cells/well) with or without 3.34 µg/mL CSPGs for 48 h, then 20 μL hydrogels (CAQK-LIP-GFs@HP, CAQK-LIP-DTX@HP, CAQK-LIP-GFs/DTX@HP; containing 5 μg/mL aFGF/BDNF and/or 50 μg/mL DTX) were added into the transwell inserts (pore size 0.4 mm) and co-incubated for 72 h. After that (DIV5, 5 days in vitro), cells were fixed with paraformaldehyde (4%) and prepared for immunofluorescent staining, which will be described later.

Wang Q., Zhang H., Xu H., Zhao Y., Li Z., Li J., Wang H., Zhuge D., Guo X., Xu H., Jones S., Li X., Jia X, & Xiao J. (2018). Novel multi-drug delivery hydrogel using scar-homing liposomes improves spinal cord injury repair. Theranostics, 8(16), 4429-4446.

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Isolation and Culture of Pre-Conditioned DRG Cells

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For pre-conditioned DRG cultures, ipsilateral L4/L5 DRGs were collected 5 days after a sciatic nerve crush. Appropriate L4-5 DRGs from five mice (naïve or pre-conditioned) were pooled and processed in one tube per experiment as in (Ma et al., 2011 (link); Seijffers et al., 2007 (link)). Once dissociated, 3,000 cells were grown per chamber in an 8-well slide, coated with myelin (100ng/well) or CSPGs (20ng/well, Millipore) and 1,500 cells were grown on laminin (5μg/well, Invitrogen).

Omura T., Omura K., Tedeschi A., Riva P., Painter M.W., Rojas L., Martin J., Lisi V., Huebner E.A., Latremoliere A., Yin Y., Barrett L., Singh B., Lee S., Crisman T., Gao F., Li S., Kapur K., Geschwind D.H., Kosik K.S., Coppola G., He Z., Carmichael S.T., Benowitz L.I., Costigan M, & Woolf C.J. (2015). Robust axonal regeneration occurs in the injured CAST/Ei mouse central nervous system. Neuron, 86(5), 1215-1227.

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Investigating Neuronal Cell Adhesion and Neurite Outgrowth

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Coverslips in 24-well plates were first coated with 200 μg/mL PLL (Sigma) for 2 hours at 37°C. After three washes with sterile water, the coverslips were coated with 1μg/mL of CSPGs (Millipore, Temeula, CA, USA) diluted in PBS or PBS only; alternately, the coverslips were coated with 5 μg/mL Laminin (LN) (Thermo Fisher) with or without 1 μg/mL CSPGs diluted in PBS. Dissociated CGNs were seeded onto the coated coverslips at a density of 1 × 10⁴ cells/well. Cells were allowed to adhere to coverslips for 30 minutes and 2 hours, and were then directly fixed with 4% PFA in the wells after gently removing culture medium without a pre-wash with PBS. Cells were stained with Texas Red-Phalloidin and DAPI. The area of individual cells at 30 minutes after plating on different substrates was measured using ImageJ. The percentage of cells with neurites longer than two cell body size was calculated at 2 hours after plating. Data were collected from three independent experiments.

Jin J., Tilve S., Huang Z., Zhou L., Geller H.M, & Yu P. (2018). Effect of chondroitin sulfate proteoglycans on neuronal cell adhesion, spreading and neurite growth in culture. Neural Regeneration Research, 13(2), 289-297.

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Chondroitinase Activity Quantification

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293T cells (ATCC) were plated at 90% confluency in 6 well plates (Cellstar) and transfected with Lenti-Chase or EGFP control plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions with 2.4μg DNA and 3μl Lipofectamine per well. After two days of culture at 37°C in a 5% CO2 incubator in 2ml Optimem media (Gibco), media was collected for chondroitinase activity measurement. The Proteoglycan Detection Kit (Amsbio com) quantifies sulfated glycoaminoglycans using 1,9 – dimethylmethylene (DMMB) dye, which shifts it’s absorption spectrum upon GAG binding. To generate a standard curve of chondroitinase activity, 0.5μg of CSPGs (Millipore) were incubated with chondroitinase enzyme (Amsbio) at 0, 12.5, 25, 50, and 100mU/ml for 2 hours at 37°C, exposed briefly to DMMB, and then 525nM absorbance as quantified by microplate reader (Molecular Devices). To quantify chondroitinase activity generated by transfected cells, 0.5μg of CSPGs were incubated with a 1:1000 dilution of conditioned media from 293T cells transfected or transduced with chondroitinase constructs or mCherry control. All test and standard curve controls were run in duplicate.

Wang Z., Winsor K., Neinhaus C., Hess E, & Blackmore M.G. (2016). Combined chondroitinase and KLF7 expression reduce net retraction of sensory and CST axons from sites of spinal injury. Neurobiology of disease, 99, 24-35.

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