Frozen tissues were first weighted before the incubation with acetone to remove the lipids. The samples were then dried and cut into small pieces before the pronase treatment (15 mg pronase per hemisphere, (Roche, cat. no. 11459643001) in 0.1 M Trizma hydrochloride (Sigma-Aldrich, cat. no. T3253), 10mM calcium acetate (Millipore, cat. no. 567418), pH 7.8. Samples were homogenized with Potter Elvehjem tissue homogenizer in the pronase solution. Residual protein fragments were precipitated with trichloroacetic acid (Sigma-Aldrich, cat. no. T6399). The supernatant, which contains the GAGs, was collected and stored on ice. The solution was then neutralised with 1 M Na2CO3 (Sigma-Aldrich, cat. no. S7795) to pH 7.0, and the GAGs were recovered by ethanol precipitation. The isolated GAGs were redissolved in 0.3 ml of deionised water. The GAG concentration was quantified using cetylpyridium chloride (CPC) turbidimetry. Standard curve was prepared from 1µg/µl of Chondroitin sulphate A (Sigma-Aldrich, cat. no. C9819)31 . Briefly, the diluted sample was mixed with 0.2% (w/v) CPC and with 133 mM MgCl2 (Sigma-Aldrich, cat. no. M2670) in ratio 1:1. Absorbance was measured at 405 nm using plate reader spectrophotometer (FLUOstar® Omega, BMG LABTECH). Each sample was carried out in three different dilutions and each dilution was carried out in duplicate.
Chondroitin sulphate a
Manufactured by Merck Group
Sourced in United States
Chondroitin sulphate A is a chemical compound derived from cartilage tissue. It is a structural component of cartilage and other connective tissues. Chondroitin sulphate A is commonly used in laboratory research applications.
Automatically generated - may contain errors
Lab products found in correlation
Heparin, by Merck Group (4 mentions)
Ifn λ1, by Thermo Fisher Scientific (3 mentions)
Ifn β, by Thermo Fisher Scientific (3 mentions)
Heparan sulfate from bovine kidney, by Merck Group (3 mentions)
Tnf β, by Thermo Fisher Scientific (3 mentions)
Chondroitin sulphate b, by Merck Group (3 mentions)
Ifn ω, by Thermo Fisher Scientific (3 mentions)
Ifn γ, by Thermo Fisher Scientific (3 mentions)
Il 18bp fc, by Thermo Fisher Scientific (3 mentions)
Chondroitin sulphate c, by Merck Group (2 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
Na2co3, by Merck Group (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Trizma hydrochloride, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Heparin binding plates, by BD (1 mentions)
Heparin sulphate, by Merck Group (1 mentions)
Tissue lysis buffer, by Merck Group (1 mentions)
10 protocols using chondroitin sulphate a
1
Quantification of Glycosaminoglycans in Tissues
2
Purification and Characterization of ps20 Protein
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Using an Aktӓ-purifier FPLC system (GE Healthcare). 5 ml of 293T-ps20 CM was absorbed to a 1 ml HiTrap™ heparin-coated column (GE Healthcare) followed by 5 CV of washing with 50 mM Tris–HCl buffer (pH 7.5). A 0–0.5 M NaCl gradient was then applied to the column over 10 CV while 0.5 ml fractions were collected. These were then subjected to WB with 1G7. Heparin binding plates (BD Biosciences) coated overnight with 25 μg/ml GAGs; heparin sulphate, chondroitin sulphate A, and chondroitin sulphate C (all Sigma Aldrich) and blocked in PBS with 1% BSA (w/v). rps20293F diluted in PBS was absorbed for 2 h prior to washing and detection with 1G7-HRP. 100 μl of tetramethylbenzidine ELISA substrate was added and absorbance at 450 nm was measured as described for the ps20 ELISA above. A baseline was established by binding of rps20293F to a BSA solid phase. For the cell binding assay 293T cells, pre-treated with sodium chlorate where indicated, were resuspended using PBS (Gibco) with 4 mM EDTA (Sigma Aldrich). 106 cells were incubated with rps20V5+/− heparin with agitation for 1 h. Cells were washed once with 1 ml PBS and resuspended in 50 μl tissue lysis buffer (Sigma). Samples were then subjected to WB as indicated.
3
Chondroitin Sulfate Phantom Preparation
4
GAG-binding affinity of rIxsS17
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Secondary structure modeling predicted at least one basic patch in IxsS17 comparative modeling structure. To test if the rIxsS17 basic patch is functional, rIxsS17 binding affinity of glycosaminoglycans (GAGs): heparin (Sigma-Aldrich, MO, USA), chondroitin sulphate A (Sigma-Aldrich), heparan sulphate (Galen Laboratory Supplies, North Haven, CT) and dermatan sulphate (Galen Laboratory Supplies) was done as previously described [48 (link)]. A GAG-binding microplate (Galen Laboratory Supplies) was coated with 200 μL of GAG at the concentration of 25 μg/mL in binding buffer (100 mM NaCl, 50 mM Na-acetate, 0.2% Tween, pH 7.2) and incubated overnight at room temperature. After washing with binding buffer, the plate was blocked with 250 μL of 1% bovine serum albumin in PBS for 1 h at 37°C. Thereafter, different concentrations of rIxsS17 (0, 1, 2, 5, 10 and 20 μg/mL) in 200μL of blocking buffer was added and incubated for 2 h at 37°C. After the wash, 200 μL of HRP-conjugated anti-histidine antibody (1:5,000 dilution) was added. Following by addition of 200 μL of 1-step Ultra TMB ELISA substrate (Thermo Scientific), 100 μL of hydrochloric acid (1N) was used to stop the reaction; and the OD450 nm was determined using a microplate reader (Biotek Synergy H1).
5
Chondroitin Sulfate A Binding Assay
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Ibidi μ‐Slide 0.2 channel slides were incubated with 100 μl chondroitin sulphate A (100 μg/ml; Sigma Aldrich) in 1× PBS overnight at 37°C. Channels were blocked with 1% (w/v) BSA–PBS for 1 h at room temperature and flushed with warm bicarbonate‐free RPMI. CS2 parasites panned against chondroitin sulphate A98 were synchronized to 15–20 hpi, drug treated for 5 h to 20–25 hpi and diluted to 3% parasitaemia and 1% haematocrit in bicarbonate‐free RPMI 1640. Cells were passaged through the channel at 100 μl/min for 10 min at 37°C. Unbound cells were washed from the channel at 100 μl/min for 10 min at 37°C. Bound cells were imaged by widefield microscopy using a Zeiss Cell Observer widefield fluorescence microscope and counted over 10 fields of view (281.28 × 178.14 μm each) using ImageJ.
6
Quantitative Chondroitin Sulfate Assay
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DMMB (dimethylenmethylblue, Sigma-Aldrich) specifically stains chondroitin sulphate. Ten microlitres of the respective eluted GAG sample or of a standard dilution series (1:10) of known concentrations of chondroitin sulphate A (Sigma-Aldrich), used for calibration, were mixed with 190 μL DMMB staining solution (10 mL 2 M NaCl, 1.5 g glycine solved in 490 mL H2O, pH 3.0 adjusted with HCl; 8 mg DMMB solved in 2.5 mL ethanol and added) and measured in an ELISA-reader (Multiscan Go, Thermo Fisher Scientific) at 520 nm. All measurements were performed in triplets.
7
Glycosaminoglycan Content Quantification
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Glycosaminoglycan (GAG) content was evaluated by the dimethylmethylene blue assay (DMMB) (Sigma, 341088). Chondroitin sulphate A (Sigma, C9819) was used to generate 6 standards, concentrations ranging from 0 to 50 µg/mL Chondroitin sulphate C (Sigma, C4384) was used to generate low and high Internal Quality Control (IQC) solutions, concentrations 15 and 35 µg/mL, respectively. Cartibeads were digested with proteinase K (1 mg/mL, Promega, V3021) in Tris-HCl buffer (50 mM, pH 8, Sigma), for 15 ± 2 h at 56 °C. The enzymatic digestion was stopped by incubation at 97 °C for 15 minutes. The resulting sample was diluted (1:5-1:10) in Tris-HCl buffer (50 mM, pH 8) for the assay. One hundred micro liters of standard, ICQ, or sample were read in triplicates with a spectrophotometer (λ = 525 nm) after 5 minutes reaction with 1 mL of DMMB working solution.
8
Recombinant Cytokines and Glycosaminoglycans
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Recombinant human cytokines used in this study (CCL1, CCL2, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12α, CXCL12β, CXCL13, CXCL14, CXCL16, XCL1, CX3CL1, IL-1α, IL-1β, IL-6, IL-6Rα, IL-10, IL-12p70, IL-13, IL-17a, IL-18BP-Fc, IL-23, IL-27, IL-35, TNF-α, TNF-β, IFN-β, IFN-γ, IFN-λ1, IFN-ω) from PeproTech, and (IFN-α2 and IL-18) from Sino Biological, were reconstituted in DPBS 0.1% BSA at 10 μM, aliquoted and stored at −80° C. Heparin (# 2106), heparan sulfate from bovine kidney (# H7640), chondroitin sulphate A (# C9819) and chondroitin sulphate B (# C3788) were obtained from MilliporeSigma. Heparan sulfate from porcine mucosa (# AMS.GAG-HS01) and keratan sulfate (# AMS.CSR-NAKPS2-SHC-1) were purchased from ASMBIO. We assumed an average molecular weight of 30 kDa for heparan sulfate from porcine mucosa and 15 kDa for Heparin36 .
9
Cytokine and Glycosaminoglycan Characterization
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Recombinant human cytokines used in this study (CCL1, CCL2, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12α, CXCL12β, CXCL13, CXCL14, CXCL16, XCL1, CX3CL1, IL-1α, IL-1β, IL-6, IL- 6Rα, IL-10, IL-12p70, IL-13, IL-17a, IL-18BP-Fc, IL-23, IL-27, IL-35, TNF-α, TNF-β, IFN-β, IFN-γ, IFN-λ1, IFN-ω) from PeproTech, and (IFN-α2 and IL-18) from Sino Biological, were reconstituted in DPBS 0.1% BSA at 10 µM, aliquoted and stored at −80° C. Heparin (# 2106), heparan sulfate from bovine kidney (# H7640), chondroitin sulphate A (# C9819) and chondroitin sulphate B (# C3788) were obtained from MilliporeSigma. Heparan sulfate from porcine mucosa (# AMS.GAG-HS01) and keratan sulfate (# AMS.CSR-NAKPS2-SHC-1) were purchased from ASMBIO. We assumed an average molecular weight of 30 kDa for heparan sulfate from porcine mucosa and 15 kDa for Heparin 36 .
10
Cytokine Reconstitution and Characterization
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Recombinant human CKs used in this study (CCL1, CCL2, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12α, CXCL12β, CXCL13, CXCL14, CXCL16, XCL1, CX3CL1, IL-1α, IL-1β, IL-6, IL-6Rα, IL-10, IL-12p70, IL-13, IL-17a, IL-18BP-Fc, IL-23, IL-27, IL-35, TNF-α, TNF-β, IFN-β, IFN-γ, IFN-λ1, IFN-ω) from PeproTech, and (IFN-α2 and IL-18) from Sino Biological, were reconstituted in DPBS 0.1% BSA at 10 μM, aliquoted and stored at −80° C. Heparin (# 2106), heparan sulfate from bovine kidney (# H7640), chondroitin sulphate A (# C9819) and chondroitin sulphate B (# C3788) were obtained from MilliporeSigma. Heparan sulfate from porcine mucosa (# AMS.GAG-HS01) and keratan sulfate (# AMS.CSR-NAKPS2-SHC-1) were purchased from ASMBIO. We assumed an average molecular weight of 30 kDa for heparan sulfate from porcine mucosa and 15 kDa for Heparin (65 ).
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