FITC was covalently conjugated to NEP1-40 and ChABC (Sigma). To fluorescently label the molecules, a solution of 2 mg/mL ChABC or 2 mg/mL NEP1-40 was dissolved in 0.1 M sodium carbonate buffer, pH 9. FITC (Sigma) was freshly dissolved in dimethyl sulfoxide (DMSO) at 1 mg/mL, and 50 µL of the FITC solution was then slowly added to the ChABC/NEP1-40 solution in 5 µL aliquots while continually stirring. The solution was allowed to react for 8 hours at 4°C and protected from light. Ammonium chloride was added to a final concentration of 50 mM and incubated for 2 hours to stop the reaction. The finished reaction was then dialyzed against phosphate buffered saline, pH 7.4, to remove unconjugated FITC. The molecular weight cutoff for the ChABC dialysis cassette was 10 kD and the cutoff for the NEP1-40 dialysis cassette was 2 kD. Following dialysis, the conjugates were stored at 4°C. The ratio of fluorescein to ChABC/NEP1-40 was determined by measuring the absorbance at 495 nm and 280 nm. Electrophoresis with polyacrylamide gels was also used to verify the overlap of fluorescent ChABC protein bands with Coomassie Blue stained bands.
Chabc
Manufactured by Merck Group
Sourced in Germany, United States
ChABC is a laboratory enzyme product manufactured by Merck Group. It is a recombinant chondroitinase ABC enzyme used in research applications.
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Lab products found in correlation
Maxq 4000, by Thermo Fisher Scientific (2 mentions)
Pnase, by Merck Group (2 mentions)
Hase 1 3, by Merck Group (2 mentions)
Hase 2, by Merck Group (2 mentions)
Dmi8 widefield microscope, by Leica (2 mentions)
Aggrecan, by Merck Group (2 mentions)
P ase, by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Nep1 40, by Merck Group (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Hyase, by Merck Group (1 mentions)
Rabbit anti parvalbumin, by Abcam (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Biotinylated wfa, by Merck Group (1 mentions)
Rabbit anti myc tag, by OriGene (1 mentions)
Chicken anti neun, by Merck Group (1 mentions)
Mouse anti 6x his tag, by Abcam (1 mentions)
Caco 2, by Thermo Fisher Scientific (1 mentions)
Hct116, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
40 protocols using chabc
1
Fluorescent Labeling of ChABC and NEP1-40
2
Enzymatic Extracellular Matrix Digestion
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We followed an established approach for enzymatic ECM digestion [7 (link), 54 (link), 56 (link)]. The treatment solutions were labelled in a non-descriptive manner (i.e. A, B, C), making the experimenter blinded during the treatment, data acquisition, and data analysis. Cell cultures were incubated with 500 mU/ml ChABC (C3667, Sigma-Aldrich, Taufkirchen, Germany) or 500 U/ml hyaluronidase (HYase, H4272, Sigma-Aldrich) for 16 h. For ECM depletion in vivo, 500 mU ChABC or 500 U HYase were dissolved in 2 µl of 0.1 M PBS and delivered via a single stereotactic (bregma 0, left 3 mm, deep 1 mm) intracortical injection. At 16 h post-injection, animals were sacrificed and brains were processed for further analysis. Control animals were treated with a vehicle (0.1 M PBS). In our hands, the two enzymes were equally efficient for ECM digestion and specifically targeted the neuronal cell culture compartment, while no alterations in glial cells were detected (Fig. S3). Moreover, ChABC and HYase induced identical changes in synapse density and network activity (Fig. S4).
3
Chondroitinase Delivery in Fibrin Scaffold
4
Neural Stem Cell Migration on CSPG Substrates
5
SARS-CoV-2 Attachment Inhibition Assay
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VeroE6, A549, and Calu-3 cells were treated by DMEM, 4H30 (12.5 μg/mL), BSA, CS, HS (5 μg/mL), 4H30 + BSA, 4H30 + CS, or 4H30 + HS at 37 °C for 30 min and then washed by PBS. SARS-CoV-2 (MOI = 0.2) was added to cells for attachment at 4 °C for 1 h. After washing the unbound virus, the attached virus was measured by RT-qPCR. For ChABC and Hase assay, A549 cells were treated by ChABC (Sigma, Cat# C2905, 1 U/mL), Hase I-III (Sigma, Cat# H3917, 100 mU/mL), and Hase II (Sigma, Cat# H6515, 100 mU/mL) at 37 °C for 2 h in the buffer according to the manufacture introduction. After washing cells with PBS, DMEM or 4H30 (12.5 μg/mL) was added to cells for 30 min incubation and then cells were washed with PBS. SARS-CoV-2 (MOI = 0.2) was added to cells for attachment at 4 °C for 1 h. The unbound virus was washed by PBS. The attached virus was measured by RT-qPCR.
6
Neural Stem Cell Migration on CSPG Substrates
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To determine the effects of CSPGs on the migration of neural stem
cells in vitro, flat-bottom 48-well plates were coated with
poly-L-lysine and various concentrations (1ug/mL and 10ug/mL) of Aggrecan
(A1960, sigma). The control wells contained poly-L-lysine alone.
Neurospheres of similar size were seeded in each well in NBM-GF medium with
2.5μM ISP peptide or scrambled peptide (n = 7 neurospheres per
condition), and the plates were incubated at 37°C for 21 hours.
Thereafter, images of each well were taken using a Leica DMi8 widefield
microscope. The Migration Index was defined as dividing the total area of
migrated cells by the inner area of neurospheres. The inner area and total
area of neurospheres were measured using ImageJ software. Time lapse
analyses (duration 21 hours) of the migration process (Video S1 , Supplementary data ) was taken
using a Leica DMi8 widefield microscope equipped with an on-stage incubator.
For application of signaling pathway inhibitors, neurospheres were treated
with LY294002 (10μM, AKT inhibition), PD98059 (10μM, ERK
inhibition) or OA-Hy (100nM, MMP2 inhibition) for 30min followed by exposure
to 2.5μM ISP peptide or scrambled peptide. For ChABC treatment,
neurospheres were incubated with 5 mU/mL ChABC (Sigma, # C2905) for 21 hours
and then NSCs migration was measured. Each experiment was repeated twice
with similar results and representative results are presented.
cells in vitro, flat-bottom 48-well plates were coated with
poly-L-lysine and various concentrations (1ug/mL and 10ug/mL) of Aggrecan
(A1960, sigma). The control wells contained poly-L-lysine alone.
Neurospheres of similar size were seeded in each well in NBM-GF medium with
2.5μM ISP peptide or scrambled peptide (n = 7 neurospheres per
condition), and the plates were incubated at 37°C for 21 hours.
Thereafter, images of each well were taken using a Leica DMi8 widefield
microscope. The Migration Index was defined as dividing the total area of
migrated cells by the inner area of neurospheres. The inner area and total
area of neurospheres were measured using ImageJ software. Time lapse
analyses (duration 21 hours) of the migration process (
using a Leica DMi8 widefield microscope equipped with an on-stage incubator.
For application of signaling pathway inhibitors, neurospheres were treated
with LY294002 (10μM, AKT inhibition), PD98059 (10μM, ERK
inhibition) or OA-Hy (100nM, MMP2 inhibition) for 30min followed by exposure
to 2.5μM ISP peptide or scrambled peptide. For ChABC treatment,
neurospheres were incubated with 5 mU/mL ChABC (Sigma, # C2905) for 21 hours
and then NSCs migration was measured. Each experiment was repeated twice
with similar results and representative results are presented.
7
Chondroitin Sulfate Quantification in Brain Tissue
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CS/DS disaccharides were isolated and quantified according to our previously published methods16 (link). MBH sections were isolated from 30 μm-thick PFA-fixed brain sections restricted to −1.6 to −4.2 mm from Bregma. Fixed hypothalamic sections were washed 3 x in Optima LC/MS-grade water and 1 x in 50 mM ammonium bicarbonate (pH 7.6) at room temperature (RT). Chondroitinase ABC digestion of CS/DS-GAGs was performed using 500 mU/ mL of ChABC (C3667, Sigma-Aldrich, St. Louis, MO) in 50 mM ammonium bicarbonate (pH 7.6) in a Thermo Scientific MaxQ4000 orbital shaker at 80 rpm, 37°C, for 24 h. Supernatants were collected in sterile 1.7 mL microcentrifuges tubes and spun for 10 min at 14 k x g to pellet any debris. The supernatant was then dehydrated using a SpeedVac Concentrator and the lyophilized product was reconstituted in 30 μL of LC/MS-grade water.
8
Chondroitinase ABC Enzymatic Digestion of PNNs
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Immediately after slicing, brain slices were incubated in 0.2 U/ml ChABC (catalog #2905, Sigma-Aldrich) or control enzyme P-ase (catalog #P0389, Sigma-Aldrich) in recording ACSF for 1 h at 35°C in a small slice chamber (BSK2, Scientific Systems Design). ChABC is a well characterized enzyme that degrades PNNs by removing glycosaminoglycan (GAG) side chains from chondroitin sulfate proteoglycans, reduces PNN labeling in acute slices (Bukalo et al., 2001 (link)), and reduces cell surface charge (Morawski et al., 2015 (link)).
9
NCAM-EphA3 Signaling Modulation
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HEK293T cells were transfected with NCAM-140 and EphA3. Recombinant neurocan was treated with 0.1 U/microgram chABC (Sigma) at 37 °C for 1.5 hr or incubated with no enzyme (as a control), followed by heat inactivation of enzyme at 100 °C for 10 min. Efficacy of chABC treatment was assessed by immunoblotting. Cells were treated with neurocan (20 nM), chABC treated neurocan (20 nM), or no neurocan (control) for 30 min. Cells were lysed and NCAM immunoprecipitated, and bound EphA3 and neurocan were detected by immunoblotting.
10
Isolation and Characterization of PBMCs
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PBMCs were isolated from the blood of healthy donors (REC 17/LO/1061). They were collected between the top plasma layer and the bottom ficoll medium of a Histopaque1077 (Sigma-Aldrich) gradient and resuspended in PBS, washed and plated in a petri dish in RPMI 1640 + 10% FBS incubated 45 min to deplete macrophages. The supernatant was then harvested and PBMC were counted and frozen in FBS 90% and DMSO 10%. For ChABC (Sigma) treatment, the compound was added to cell culture media at the final concentration of 0.01U/ml for 48 h. Then cells were stained for chondroitin 56 (Abcam) or harvested to be plated in transwell chamber.
Conditioned-media from GICs or iNSCs or cells (20 K) were added to the geltrex coated top chamber of a transwell 5 μm pore (Sarstedt, 83.3932.500) on a 24 wells plate. Twenty-four hours later, PBMCs (300 K cells) in RPMI with FBS 10%, were added to the top chamber for a 4 h incubation at 37 C, CO2 5%. Cells were harvested in the bottom chamber, counted, and stained by flow cytometry.
Conditioned-media from GICs or iNSCs or cells (20 K) were added to the geltrex coated top chamber of a transwell 5 μm pore (Sarstedt, 83.3932.500) on a 24 wells plate. Twenty-four hours later, PBMCs (300 K cells) in RPMI with FBS 10%, were added to the top chamber for a 4 h incubation at 37 C, CO2 5%. Cells were harvested in the bottom chamber, counted, and stained by flow cytometry.
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