Hydroxyproline Assay (UUH protocol, Update Mar )

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1 Hydroxyproline Assay (UUH protocol, Update Mar ) Euthanized mouse with CO 2 (or i.v. anesthesia with neck bone dislocation) and perfuse lungs with 1xPBS, removed lung and placed in a 5 ml tube and snap freeze in Liquid Nitrogen and stored in the -80 C or used right away Homogenize the lungs in 1 ml PBS with complete tab; homogenizer is washed out in 3 vials PBS between treatment groups or every sample. Remove residual connective tissue from homogenizer as well. Transfer 500 μl of homogenate to glass tubes with screw on caps (Fisher Science, Pyrex VISTA Reusable Glass Tubes with Phenolic Screw Caps Corning No.: ) and add 500 μl of 12N HCl (35-38% original solution). Screw caps on tightly or solution will boil out. Put tube in 120 C oven for minimum of 8 hours to overnight. Filter the ashes in tubes through Glass wool filters (Sigma-Aldrich, Whatman quantitative filter paper, hardened ashless, Grade 540 circles, diam. 55 mm, Cat. No Z241539) from glass tube to 1.5 ml eppendorf tube Make up Chlotamine T solution fresh (stock into the refrigerator (4 ºC) and 3 days after, make a new solution) (If the samples are dried completely, add 500 μl of dh 2 O (Distilled Water) into the each tube) Add 5 μl sample to each well of 96-well flat bottom plate Samples should be run neat (and 1:2 if needed) load 12 standards as well. Add 5 μl of Citrate/Acetate buffer and tap side of plate to mix Add 100 μl of Chloramine T solution to all wells tap to mix again Turn on oven: 65 C, Incubate samples at RT for 20 min Add 100 μl of Ehrlich s solution to each well and incubate at 65 C for minutes or until; your standards have changed color Read plate at 550 nm Hydroxyproline standard stock 10 mg Hydroxyproline (Sigma-Aldrich H5534) in MQ (Milli-Q) (deionized filtered water) Standard: First dilute stock (4mg/mL) 1:10; 400 μg/ml-0 μg/ml (dilute 1:2 in sterile water); 5 μl of 1 / 8

2 standard is loaded per well 10 mg/vial ml =2.5 ml of 4 mg/ml (made stock) 1 10 μl of 4mg/ml Hydroxyproline Standard Solution with 90 μl of water (MQ) to prepare a 0.4 mg/ml (400 μg/ml) standard solution. (100 μl) each 5 μl into 96 well 2 50 μl from μl of MQ = 100 μl of 200 μg/ml 3 50 μl from μl of MQ = 100 μl of 100 μg/ml 4 50 μl from μl of MQ = 100 μl of 50 μg/ml 5 50 μl from μl of MQ = 100 μl of 25 μg/ml 6 50 μl from μl of MQ = 100 μl of 12.5 μg/ml 7 50 μl from μl of MQ = 100 μl of 6.25 μg/ml 8 50 μl from μl of MQ = 100 μl of 3.12 μg/ml 9 50 μl from μl of MQ = 100 μl of 1.56 μg/ml μl from μl of MQ = 100 μl of 0.78 μg/ml 11 5 μl of MQ (blank) If you use the 50mg/mL vial: dilute 1: 100 first 500 μg μg (Example) Dilute 10 μl of the 1 mg/ml Hydroxyproline Standard Solution with 90 μl of water to prepare a 0.1 mg/ml standard solution. Add 0, 2, 4, 6, 8, and 10 μl of the 0.1 mg/ml hydroxyproline standard solution into a 96 well plate, generating 0 (blank), 0.2, 0.4, 0.6, 0.8, and 1.0 μg/well standards. NaOH (Sodium Hydroxide)-solution 4 M solution (40 gram/mol): 16 gram NaOH in 100 ml MQ 2 / 8

3 Reagents: Citrate/Acetate Buffer 5% Citric Acid (5g) (Sigma-Aldrich C1909, monohydrate) 1.2% Glacial Acetic Acid 1.2 ml (fw 60.05) (Fisher Scientific A38-500) 7.24% Sodium Acetate (7.24g) (Sigma-Aldrich S2889) 3.5% Sodium Hydroxide (3.4g) (Sigma-Aldrich ) Made up in 100 ml sterile water Chloramine T 0.282g Chloramine T (Sigma-Aldrich ) 2mL 2mL 16mL Iso-propranol (Fisher Scientific NC ) Sterile Water (Milli-Q is O.K) Citrate Acetate Buffer Heat in 37 C waterbath until completely dissolved Ehrlich s Solution (Sigma-Aldrich, ml, buying is easier than making) 2.5g 4-DMAB Ehrlich s Reagent (p-dimethylaminobenzaldehyde) 9.3mL Iso-Propranol 3.9mL 70% Perchloric Acid (add last and slowly) 3 / 8

4 (For Reference 2 nd protocol) Hydroxyproline Assay (Michigan Univ.) Euthanized mouse with CO2 and perfuse lungs with 1xPBS, removed lung and placed in a 5 ml tube and snap freeze in liquid nitrogen and stored in the -80 C or used right away Homogenize the lungs in 1 ml PBS with complete tab; homogenizer is washed out in 3 vials PBS between treatment groups or every sample. Remove residual connective tissue from homogenizer as well. Transfer 500 μl of homogenate to glass vials with screw on caps and add 500 μl of 12N HCL. Screw caps on tightly or solution will boil out. Put tube in 120 C oven for minimum of 8 hours to overnight. Make up Chlotamine T solution fresh Add 5 μl sample to each well of 96-well flat bottom plate Samples should be run neat (and 1:2 if needed) load 12 standards as well. Add 5 μl of Citrate/Acetate buffer and tap side of plate to mix Add 100 μlof Chloramine T solution to all wells tap to mix again Turn on oven: 65 C, Incubate samples at RT for 20 min, make up Ehrlich s Solution Add 100 μl of Ehrlich s solution to each well and incubate at 65 C for minutes or until; your standards have changed color Read plate at 550 nm Standard: First dilute stock (4mg/mL) 1:10; 400 μg/ml-0 μg/ml (dilute 1:2 in sterile water); 5 μl of standard is loaded per well If you use the 50mg/mL vial: dilute 1: 100 first 500 μg μg Reagents: 4 / 8

5 Citrate/Acetate Buffer 5% Citric Acid (5g) (C1909 Sigma, monohydrate) 1.2% Glcial Acetic Acid 1.2 ml (fw 60.05) 7.24% Sodium Acetate (7.24g) 3.5% Sodium Hydroxide (3.4g) Made up in 100 ml sterile water Chloramine T 0.282g Chloramine T 2mL 2mL Iso-propranol Sterile Water 16mL Citrate Acetate Buffer Heat in 37 C waterbath until completely dissolved Ehrlich s Solution 2.5g 4-DMAB Ehrlich s Reagent (p-dimethylaminobenzaldehyde) 9.3mL Iso-Propranol 3.9mL 70% Perchloric Acid (add last and slowly) 5 / 8

6 Michigan Univ. protocol from Red Journal : Hydroxyproline quantification. Whole lungs were homogenized in 2 ml of 0.5 M glacial acetic acid: 1 ml was processed for hydroxyproline assay, and 1 ml was used for Sircol assay. Lung homogenate was dried under vacuum for 16 h, suspended in 1 ml of 6 N HCl, and heated for 8 h at 120 C. The total digest was filtered through a 0.2-μm filter, and 50 μl of clear filtrate were dried for 16 h using a Speed Vac and then suspended in 50 μl of citrate-acetate buffer (ph 6.0) to which 1 ml of chloramine-t solution was added. 6 / 8

7 The resulting mixture was then incubated at room temperature for 20 min before 1 ml of Ehrlich solution (Aldrich, Milwaukee, WI) was added. These samples were incubated for 20 min at 65 C; after they were cooled, the samples were read at 550 nm in a spectrophotometer (model DU 640, Beckman). Hydroxyproline concentrations were calculated from a standard curve of hydroxyproline (0 100 mg/ml). Sircol assay for collagen in whole lungs. One milliliter of lung homogenate prepared as described for the hydroxyproline assay was mixed overnight at 4 C. After centrifugation, 100 μl of supernatant were mixed with 1 ml of Sircol assay dye reagent and incubated for 30 min at room temperature. After centrifugation, the pellet was suspended in 1 ml of alkali reagent and vortexed to release the dye into solution. Then 100 μl of solution were transferred to a microplate, and absorbance was measured at 540 nm. Values for experimental samples were based on a standard curve of known concentrations of purified rat tail collagen. (Am J Physiol Lung Cell Mol Physiol May;294(5):L Epub 2007 Dec 27.Michigan univ ) 7 / 8

8 (For reference: 3rd protocol: Dr. Kwon s Lab) a. Thaw lungs in 1ml water, homogenize in Fisher glass tubes (#14961). b. Add 125 µl 50% TCA (trichloroacetic acid T6399-sigma) to the homogenate and incubate on ice for 20minutes. c. Spin samples at 1000 rpm, 5min., 4. Supernatant is discarded and 1ml 12N HCl in added to the pellet in glass tube. Bake at 110 for 24h (in a glass beaker) d. Reconstitute dried pellet with 2ml dh 2 O (Distilled Water). Make up 6 hydroxyproline standards (Sigma-H6002) starting from 0.25mg/ml e. In an 1.5ml eppendorf tube containing 500ul chloramine T (1.4% chloramine T in 0.5M Na Acetate and 10% isopropanol) (ⅰ) Add 200 µl sample and incubate 20min. at room temperature. (ⅱ) Add 500 µl of Ehrlich's/PDMAB (1M p-dmba<p-dimethylaminobenzaldehyde> in 70% isopropanol and 30% perchloric acid ) incubate at 65 for 15min. (ⅲ) Transfer 100 µl of final reaction solution to 96-well plate, triplicate measurement for each sample, read at 550nm 8 / 8

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