The authors have no competing financial interests.

All the experimental procedures were approved by the Institutional Review Board (Project No. CEI-2020/06/04). The AM was obtained from the Instituto de Oftalmologia Conde de Valenciana amnion bank (from deidentified human subjects), which is prepared as described by Ch&#225;vez-Garc&#237;a et al.17.
1. Preparation of the amniotic membrane extract
<ol>
	<li>Obtain 100 mg of AM from the amnion bank. 
	NOTE: According to a previous report, 50 mg of AM secretes a total of 10 ng/mL of lumican14. To obtain a higher concentration of lumican, use 100 mg of AM, equivalent to a total area of 32 cm2.</li>
	<li>If the AM is frozen, thaw it at room temperature. 
	NOTE: Perform the following procedures under a laminar flow hood class II B.</li>
	<li>Wash the AM in a Petri dish with 10 mL of sterile balanced salt solution (BSS, see Table of Materials) for 2 min.
	<ol>
		<li>Pour the BSS into a beaker.</li>
		<li>Repeat step 3 and visually confirm that glycerol medium is not present in Petri dish BSS. 
		NOTE: Repeat step 3. as needed until glycerol medium is not present in Petri dish BSS.</li>
	</ol>
	</li>
	<li>Incubate the AM with 10 mL of dispase II (1.7 IU/mL, see Table of Materials) at 37 &#176;C, 5% CO2 for 30 min. 
	NOTE: Dispase II is a neutral protease with gentle activity over epithelial cells. This enzyme effectively separates the intact epidermis from the dermis and isolates intact epithelial sheets18.</li>
	<li>After dispase incubation, perform a mechanical de-epithelialization14 with a rubber policeman (see Table of Materials). Confirm de-epithelialization by microscopy visualization. 
	NOTE: The process of de-epithelization is corroborated in an inverted microscope using 4x and 20x objectives. Visualize the tissue to exclude the presence of any cellular layer.</li>
	<li>Wash the AM in a Petri dish with 10 mL of BSS for 2 min. Pour the BSS into a beaker.</li>
	<li>Place the de-epithelialized AM (dAM) in a 2 mL microcentrifuge tube. Immerse the dAM in liquid nitrogen for 40 min.</li>
	<li>Manually ground the frozen dAM for 2-3 min in a precooled mortar at -85 &#176;C until a fine powder is obtained.</li>
	<li>In the mortar, solubilize the dAM powder with 2.5 mL of protease inhibitor solution (BSS with protease inhibitors). 
	&#8203;NOTE: Every tablet of protease inhibitor consists of the following mixture of enzymes: pancreas extract (0.02 mg/mL), thermolysin (metalloprotease) (0.0005 mg/mL), chymotrypsin (0.002 mg/mL), trypsin (0.02 mg/mL), and papain (0.33 mg/mL) (see Table of Materials).</li>
	<li>Collect the mixture with a micropipette, and clean the mortar walls with the help of a scalpel knife. Place the mixture in a 5 mL tube.</li>
	<li>Mix well with the vortex for 30 s.</li>
	<li>Homogenize the tissue mixture by centrifuging at 34 x g for 20 min at 4 &#176;C and immediately centrifuge at 3360 x g for 20 min at 4 &#176;C.</li>
	<li>The supernatant collected is the AME (Figure 1). Store 0.7 mL of each AME in different 2 mL microcentrifuge tubes for 6, 12, 20, and 33 days at the different temperature conditions of -20 &#176;C, 4 &#176;C, and room temperature (RT).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64460/64460fig01.jpg" /> 
Figure 1: Process of AME preparation and lumican concentration measurement. 100 mg of AM were incubated with dispase II at 37 &#176;C for 30 min and mechanically de-epithelialized. The de-epithelialized AM was washed and immersed in liquid nitrogen for 40 min, and then crushed until a fine powder was obtained, which was solubilized with 2.5 mL of saline buffer with protease inhibitors and centrifuged. The supernatant was collected and stored at -20 &#176;C, 4 &#176;C and RT for 6, 12, 20, and 32 days until total protein and lumican quantitation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64460/64460fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. AME protein quantification
NOTE: The quantification of total protein in the AME must be carried out immediately after obtention. Quantify proteins using Lowry protein assay and follow the manufacturer&#39;s instructions (see Table of Materials). It is recommended that all standards and samples be assayed in triplicate.
<ol>
	<li>Pipette 40 &#181;L of each sample of AME into a 96-well microplate.
	<ol>
		<li>Prepare a standard curve into the same microplate using bovine serum albumin (BSA) standard for a final BSA concentration of 0-1,500 &#181;g/mL (0, 1, 5, 25, 125, 250, 500, 750, 1,000 and 1,500 &#181;g/mL).</li>
	</ol>
	</li>
	<li>Pipette 200 &#181;L of the modified Lowry reagent to each well. Immediately mix it on a plate mixer for 30 s.</li>
	<li>Cover the microplate with aluminum foil and incubate it at RT for 10 min.</li>
	<li>Pipette 20 &#181;L of 1x Folin-Ciocalteu reagent to each well. Immediately mix it on a plate mixer for 30 s. 
	NOTE: To prepare 1x Folin-Ciocalteu reagent, dilute 2x (2N) reagent 1:1 with ultrapure water. Prepare 1x Folin-Ciocalteu reagent on the same day of use as the diluted reagent is unstable.</li>
	<li>Cover the microplate from light with aluminum foil and incubate it at RT for 30 min.</li>
	<li>Measure the absorbance of samples at 660 nm in an ELISA plate spectrometer (see Table of Materials). 
	NOTE: Color can be measured at wavelengths between 650 nm and 750 nm.</li>
	<li>Average the 660 nm absorbance value of the standard blank samples and subtract it from other 660 nm values of standard and unknown samples.
	<ol>
		<li>Measure the absorbance with an ELISA plate spectrometer in an endpoint mode with low shaking for 10 s.</li>
	</ol>
	</li>
	<li>Use the standard curve to determine the protein concentration of each unknown sample.</li>
	<li>For the calculation of protein, determine the concentration from a lineal regression graph using the values of absorbance on the Y axis against the concentrations in mg/mL on the X axis of each standard BSA curve.
	<ol>
		<li>Obtain the equation of linear regression and r value to calculate the protein concentration. 
		&#8203;NOTE: Results are expressed as normalized relative concentration values of total protein relative to mg of AM (&#181;g/mL protein/mg AM tissue).</li>
	</ol>
	</li>
</ol>
3. Quantification of Lumican in AME
NOTE: The concentration of lumican must be measured in the AME stored at different storage conditions and time periods. Quantify lumican using sandwich ELISA and follow the manufacturer&#39;s instructions. It is recommended that all standards and samples be assayed in duplicate.
<ol>
	<li>Dilute the human lumican capture antibody (see Table of Materials) to the employed concentration in phosphate-buffered saline (PBS). 
	NOTE: The capture antibody vial contains 120 &#181;g of antibody. After reconstitution with 0.5 mL of PBS, dilute the capture antibody at a working solution of 2 &#181;g/mL.
	<ol>
		<li>Instantly pipette 100 &#181;L per well of the diluted capture antibody to a 96-well microplate. Enclose the plate and incubate it overnight at RT.</li>
	</ol>
	</li>
	<li>Aspirate each well and wash it by pipetting with 300 &#181;L of wash buffer: 0.05% polyoxyethylene sorbitan monolaurate 20 in PBS, pH 7.2-7.4 (see Table of Materials) using a multi-channel pipettor. Repeat three times. 
	NOTE: After the last wash, remove any remaining wash buffer by everting the plate and gently tap it against paper towels.</li>
	<li>Block plates by adding 300 &#181;L of reagent diluent: 1% BSA in PBS, pH 7.2-7.4, 0.2 &#181;m filtered (see Table of Materials) to each well. Incubate at RT for 1 h.</li>
	<li>Repeat step 2.</li>
	<li>Prepare a standard curve into a 96-well microplate using two-fold serial dilutions from 0-8,000 pg/mL for final concentrations of 125, 250, 500, 1,000, 2,000, 4,000 and 8,000 pg/mL. The ELISA lumican kit contains a recombinant lumican standard of 75 ng (see Table of Materials).</li>
	<li>Add 100 &#181;L of samples and the standard curve in the capture antibody-coated 96-well microplate.</li>
	<li>Cover the microplate and incubate for 2 h at RT with low agitation in a compact rocker maintaining the velocity between 2-3 rpm.</li>
	<li>Repeat step 2.</li>
	<li>Add 100 &#181;L of the biotinylated-detection antibody (see Table of Materials) to each well. Cover from light and incubate 2 h at RT with low agitation in a compact rocker maintaining the velocity between 2-3 rpm. 
	NOTE: The biotinylated-detection antibody vial contains 24 &#181;g of antibody. After reconstitution with 1.0 mL of reagent diluent, dilute the biotinylated-detection antibody at a working solution of 400 ng/mL.</li>
	<li>Repeat step 2.</li>
	<li>Add 100 &#181;L of the working dilution of streptavidin-horseradish peroxidase (HRP, see Table of Materials) to each well. Cover the microplate from light and incubate for 20 min at RT. 
	NOTE: The reactive streptavidin-HRP was 40-fold concentrated. The work solution 1x of streptavidin- HRP was made with reagent diluent. 
	NOTE: Avoid placing the plate in direct light.</li>
	<li>Repeat step 2.</li>
	<li>Finally, add 100 &#181;L of substrate tetramethylbenzidine (TMB, see Table of Materials) solution to each well. 
	NOTE: Prepare TMB solution with an equal volume of stabilized hydrogen peroxide 30% solution provided in the kit. 
	NOTE: Prepare the solution immediately before use and maintain it at room temperature.</li>
	<li>Incubate for 30 min at RT in a dark place. 
	NOTE: Avoid placing the plate in direct light. Do not aspirate the TMB solution as no further washing is needed.</li>
	<li>Add 50 &#181;L of 1N H2SO4 stop solution to stop the colorimetric reaction. Gently tap the plate to ensure thorough mixing.</li>
	<li>Immediately determine the absorbance of each well using a microplate reader set to 450 nm in an ELISA plate spectrometer.</li>
	<li>Measure the absorbance with an ELISA plate spectrometer in an endpoint mode with low shaking for 10 s.</li>
	<li>Average the 450 nm absorbance value of the standard blank samples and subtract it from other 450 nm values of standard and unknown samples.</li>
	<li>Use the standard curve to determine the lumican concentration of each unknown sample.</li>
	<li>For the calculation of lumican concentration, make a lineal regression graph using the values of absorbance on the Y axis against the concentrations in pg/mL on the X axis of each standard lumican curve.
	<ol>
		<li>Obtain the equation of linear regression and r value to calculate the lumican concentration. 
		NOTE: The concentration of lumican was normalized with respect to the mg of tissue extracted. Results are expressed as normalized relative concentration values of lumican to mg AM (ng/mL lumican/mg AM tissue).</li>
	</ol>
	</li>
</ol>

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The study was funded by the Support Program for Research and Technological Innovation Projects of the Universidad Nacional Autonoma de Mexico (Grant No. PAPIIT IN203821), and Ministry of Education, Science, Technology and Innovation (Grant No. SECTEI 250/2019).

In this study, the presence of lumican was analyzed in the AME and its direct correlation with its stability under different storage conditions. Interestingly, when the total protein concentration in AME was quantified, protein concentration increased after storage. Evidence suggests three mechanisms that could change protein concentration in frozen storage: cold denaturation, the frozen concentration of solutes, and ice-induced partial unfolding of protein structure19. The freezing process could affect protein concentration on storage samples due to the crystallization of the liquid phase in the sample. The results suggest that this could have occurred as a process of freezing the concentration, affected by the storage time in the freezer. A higher concentration of proteins was observed at longer times of storage (32 and 20 days) and at the coldest temperatures (4 &#176;C and -20 &#176;C). However, the period with the highest concentration of protein did not have the highest concentration of lumican; this suggests that lumican could be affected by frozen temperatures and time conditions.
According to the results, lumican concentration was higher and more stable at 12 days of storage at 4 &#176;C and -20 &#176;C. Nonetheless, a lesser concentration of lumican was found at 6 days compared to 12 days. Some reports suggest that protein concentration could change after frozen storage conditions20. The results could be caused by a thermodynamic mechanism named ice-induced partial unfolding of protein structure, which happens during the freezing of samples. The interaction of proteins with water in the aqueous solutions reduces their interactions with other molecules. The crystallization process of water under frozen conditions allows proteins and some functional regions to interact with other molecules19. By the aforementioned, after 12 days of freezing lumican in an aqueous solution, it could be able to interact with the antibodies present in the ELISA quantification kit to result in a higher concentration. On the other hand, probably on the sixth day, the lumican could have been secluded in the aqueous solution.
In advancing innovations, the use of AM is the state-of-the-art treatment for corneal re-epithelialization regardless of etiology. As aforementioned, the benefits of AM are vast21,22,23,24,25,26. Many authors have demonstrated the benefits of lumican in AME, making it an affordable alternative specifically for developing countries1,2,3,4,5,6,7,8,9. Currently, there are many benefits of lumican in AME; its use as a treatment favors corneal re-epithelialization and improves the prognosis of corneal ulcers4,5,6,7,8,9,10,11,12,13,14,15. AM transplantation (AMT) has become a treatment with great benefits for improving various corneal disorders24,25. However, there are some chronic affections of the corneal tissue, such as persistent epithelial defects (PED) and limbal stem cell deficiencies (LESCD), that require constant treatment and maintenance of the presence of biological factors that help corneal repair21,24. Currently, there are no adjuvant treatments that allow long-term maintenance of the factors released by AMT on the corneal surface. However, a constant replacement of the AMT could not be recommended for patient safety26. For this reason, it is necessary to develop alternatives that allow the functions of AMT to assist and helps maintain the presence of factors released by AM in the corneal tissue such as lumican for a longer time, intending to favor the treatment of persistent problems of the cornea27.
Lumican is one of the factors present in AM with anti-inflammatory and antifibrotic functions, which has been reported to have functions in the corneal repair process6,7,8,9,10,11,12,13,14,15. That is why lumican suggests being a good candidate to aid in treating corneal affections; however, additional research is required to determine the efficacy of lumican in AME to achieve corneal re-epithelialization.
Lumican is a proteoglycan that has been shown to regulate the secretion of extracellular matrix compounds as collagen; also, it is involved in fibroblast activation and modulation of inflammatory cells and the angiogenesis process, having an important role in wound healing. According to the results, lumican can be extracted from AM tissue. Therapeutic applications of lumican are numerous; the use of lumican in the AME allows an attainable therapeutic option for ocular disorders4,13. The primary advantage of using AME is that it provides an easy application as a topical treatment for the ocular surface, given its aqueous composition. Likewise, other proteins with anti-inflammatory and immunoregulatory characteristics can be found within the components extracted from the AM tissue, which could present a greater benefit in treating de-epithelializing problems in the eye. For example, other anti-inflammatory factors such as TSG-6 present in the AM and cellular components have previously been reported to have immunoregulatory properties8. Hereby, a combined therapy of lumican and other extracellular matrix compounds and immunomodulatory molecules present in the AME could be useful in the re-epithelialization and wound healing process.
This method aims to demonstrate a straightforward technique for protein extraction, useful for the obtention of bountiful proteins and factors. One of the critical considerations for this method is the use of protease inhibitors, as it is fundamental for successful protein extraction as the AM is a tissue with loads of enzymatic compounds to prevent protein degradation27. Evidence has reported that using protein inhibitors together with an aqueous solution increases the extraction of other factors, such as HGF, in AM tissue26. The obtention of a fine powder after freezing and grounding the AM is necessary for the optimal obtention of AME, as this process is suitable for tissue and cellular disruption of structures needed for the extraction of cytosolic and other nuclear compounds28,29.
A few limitations of this extraction method are that the AM quantity used could not allow a high-scale extraction. This protocol allows protein extraction from a limited amount of tissue; since there is no evidence that this method allows obtaining a greater amount of protein from a larger tissue area. Troubleshooting to be considered in comparison with other extraction methods is to use a suitable concentration of protease inhibitor and incubation time, as the excess enzymes could affect proteins30 and reduce the technique&#39;s efficacy.
Part of this technique was modified from that reported by Mahbod et al.16, which describes that repeating the centrifugation and extraction process increases protein extraction. On contrary to Mahbod, the results did not report protein concentration after three cycles of centrifugation. When the total protein concentration was determined, it decreased by 80% in a second extraction and up to 97% in a third extraction. Performing only one extraction reduces the processing time and does not decrease the amount of total protein. With the preceding, the extraction method reported here requires only one centrifugation step with favorable results.
This technique could be used to extract other factors and proteins present in the AM and further, to obtain factors from other sources such as animals or vegetables. It could also have an application for obtaining proteins to carry out basic research or even the development of formulations and treatments.
These results suggest that lumican can be extracted from AM and stored for 12 days as AME under both -20 &#176;C and 4 &#176;C temperature conditions. It is important to consider its half-life to achieve the therapeutic effects of lumican as AME. Further studies are needed to determine the role of AME lumican in corneal epithelial cells and to determine the ideal dose of lumican for corneal re-epithelialization.
In conclusion, the results suggest obtaining factors such as lumican in the AME is possible. Likewise, temperature and storage time conditions influence the concentration of lumican present in the AME.

One of the most used treatments for corneal affections is amniotic membrane transplantation; however, in recent years, new proposals have emerged for using various components of amniotic tissue as alternative and adjuvant treatments. Among the most studied components of AM are those obtained from the AM extract (AME)1,2,3,4,5,6,7. AM contains multiple soluble factors such as antiangiogenic proteins, interleukins (IL), tissue inhibitors of metalloproteinases (TIMPs), anti-inflammatory proteins mediated by TSG-6 that inhibit neutrophil extracellular traps, growth factors: epidermal growth factor (EGF), transforming growth factor (TGF) (alpha and beta), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and lumican, which maintains corneal transparency by regulating collagen fibrillogenesis1,2,3,4,5,6,7,8,9.
Lumican is a small leucine-rich proteoglycan (SLRP), one of the main extracellular components of interstitial collagenase in the corneal stroma matrix, responsible for organizing collagen fibers and maintaining corneal transparency4,10,11. Proteoglycans are molecules in the extracellular matrix (ECM), which are the main ones in carrying out cell signaling and maintaining intracellular homeostasis12. ECM proteins have been reported to drive the cellular processes of proliferation, differentiation, and migration during wound healing11.
Evidence indicates the possible participation of lumican in the process of corneal re-epithelialization. Saika et al., in a study, showed that after a corneal injury, lumican could be detected in corneal keratocytes between the first 8 h and up to 3 days after injury. Presenting the highest concentration of lumican on the second and third day, this proteoglycan is subsequently undetectable on the seventh day13. These data suggest the participation of lumican in the activation of the corneal re-epithelialization process. On the other hand, in another study, it was reported that the absence of lumican delays re-epithelialization; interestingly, adding lumican could accelerate the re-epithelialization process4,11,13. Likewise, a recent study has reported that lumican can modulate the inflammatory functions of corneal limbus fibroblasts14, which suggests a role for lumican as a modulator of the inflammatory, antifibrotic and re-epithelializing response. Similarly, lumican can modulate the corneal response by interacting with signaling molecules such as Fas-FasL. Also, the absence of lumican in a knockout Lum-/- mouse model demonstrated that the lack of lumican signaling prevents adequate corneal repair15.
Primarily, this method aims to demonstrate a feasible and approachable way to extract lumican from AM. With this advantageous method of lumican extraction, it is possible to obtain similar concentrations of proteins, decreasing the processing time and making it more convenient for investigators compared to the previous studies16. Furthermore, this AME lumican could be used as an adjuvant for corneal repair and re-epithelialization processes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 N H2SO4 stop solution</td>
			<td>R&#38;D Systems</td>
			<td>DY994</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;L micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mm Petri dish</td>
			<td>Symlaboratorios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>18 G Needle (1.2 mm x 40 mm)</td>
			<td>BD Becton Dickinson&#160;</td>
			<td>305211</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>Z606340</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL plastic syringe&#160;</td>
			<td>BD Becton Dickinson&#160;</td>
			<td>302562</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;L micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20-200 &#956;L micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>30119401</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microplate</td>
			<td>SARSTEDT</td>
			<td>821581</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Amniotic membrane</td>
			<td>Instituto de Oftalmologia Conde de Valenciana Amnion Bank</td>
			<td></td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>Balanced salt solution</td>
			<td>Bausch + Lomb</td>
			<td>BSS-403802</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>BioRender&#160;</td>
			<td>BioRender</td>
			<td></td>
			<td>figures design&#160;</td>
		</tr>
		<tr>
			<td>Compact Rocker</td>
			<td>BioRad</td>
			<td>970822DD</td>
			<td>Mod. 5202SD-BIO</td>
		</tr>
		<tr>
			<td>complete, EDTA-free, Protease inhibitor 
			cocktail tablets</td>
			<td>Roche</td>
			<td>11 873 580 001</td>
			<td>Protease Inhibitor</td>
		</tr>
		<tr>
			<td>Daiggner vortex Genie 2</td>
			<td>A.Daigger &#38; Co. , INC</td>
			<td>22220A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA plate spectrometer</td>
			<td>Thermo Labsystems&#160;</td>
			<td>35401106</td>
			<td>Multiscan</td>
		</tr>
		<tr>
			<td>Freezer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism&#160;</td>
			<td>GraphPad Software, Inc</td>
			<td>version 9</td>
			<td>statistical analysis and graphic program&#160;</td>
		</tr>
		<tr>
			<td>Human lumican DuoSet ELISA kit</td>
			<td>R&#38;D Systems</td>
			<td>DY2846-05</td>
			<td>includes human Lumican capture antibody</td>
		</tr>
		<tr>
			<td>Incubator&#160;</td>
			<td>Forma Scientific&#160;</td>
			<td>3326 S/N 36481-7002</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted light Microscope</td>
			<td>Olympus&#160;</td>
			<td>6A13921</td>
			<td>to confirm de-epithelialization&#160; Mod.CK2</td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Forma Scientific&#160;</td>
			<td>14753-567</td>
			<td>Mod.1184</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mortar</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel pipettor</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen Tank</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Mod. Biocan 20</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>R&#38;D Systems</td>
			<td>DY006</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Modified Lowry Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23240</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate sealers</td>
			<td>R&#38;D Systems</td>
			<td>DY992</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent diluent</td>
			<td>R&#38;D Systems</td>
			<td>DY995</td>
			<td>1% BSA in PBS, pH 7.2-7.4, 0.2 &#956;m filtered</td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td>centurion scientific Ltd&#160;</td>
			<td>15877</td>
			<td>Mod. K2015R</td>
		</tr>
		<tr>
			<td>Rubber policeman cell scraper</td>
			<td>NEST</td>
			<td>710001</td>
			<td>for mechanical de-epithelialization</td>
		</tr>
		<tr>
			<td>Scalpel knife</td>
			<td>Braun</td>
			<td>BB521</td>
			<td>No. 10 or 21</td>
		</tr>
		<tr>
			<td>Streptavidin-HRP 40-fold concentrated&#160;</td>
			<td>R&#38;D Systems</td>
			<td>part 893975</td>
			<td></td>
		</tr>
		<tr>
			<td>Substrate tetramethylbenzidine (TMB) solution</td>
			<td>R&#38;D Systems</td>
			<td>DY999</td>
			<td></td>
		</tr>
		<tr>
			<td>Toothed tweezers</td>
			<td>Invent Germany</td>
			<td>6b</td>
			<td>inox&#160;</td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>PISA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wash buffer</td>
			<td>R&#38;D Systems</td>
			<td>WA126</td>
			<td>0.05% Tween 20 in PBS, pH 7.2-7.4</td>
		</tr>
	</tbody>
</table>

lumican extraction from amniotic membrane and determination of its storage temperature

Lumican is a small leucine-rich proteoglycan in the human amniotic membrane (AM) that promotes corneal epithelialization and the organization of collagen fibers, maintaining corneal transparency. In the present work, a method for protein extraction from AM to obtain lumican is proposed. Additionally, the stability of lumican in the AM extract (AME) stored at different temperatures and time periods is evaluated. 100 mg of AM were thawed and mechanical de-epithelialized. The de-epithelialized AM was frozen and crushed until a fine powder was obtained, which was solubilized with 2.5 mL of saline buffer with protease inhibitors and centrifuged for protein extraction. The supernatant was collected and stored at -20 &#176;C, 4 &#176;C, and room temperature (RT) for 6, 12, 20, and 32 days. Afterward, lumican was quantified in each AME. This technique allows an accessible and acquirable protocol for lumican extraction from AM. Lumican concentration was affected by storage time and temperature conditions. Lumican in the AME of 12 days stored at -20 &#176;C and 4 &#176;C was significantly higher than other AME. This lumican extraction could be useful for developing treatments and pharmaceutical solutions. Further studies are needed to determine the uses of AME lumican in re-epithelialization and wound healing process.

Results are reported as the mean value &#177; standard deviation (SD). Student&#39;s t-tests and analysis of variance (ANOVA) were performed. P-values &#60; 0.05 was considered statistically significant. Statistical analysis was performed using statistics software (see Table of Materials).
The total protein quantity in the AME was affected by time and storage conditions. The basal protein concentration was similar among all AME; the range of total protein was 2.7 &#177; 0.3 &#181;g/mL without a significant difference between the samples evaluated. However, when the samples were stored for 12, 20, and 32 days, variability in protein concentration with respect to basal concentration was observed. Interestingly, protein concentration increased in the AME at 4 &#176;C and -20 &#176;C with respect to the RT at all times of storage.
Similarly, when protein concentration was compared between storage times, it changed after 12, 20, and 32 days. A significant difference (p &#60; 0.05) was found in the AME of 32 and 20 days at 4 &#176;C and -20 &#176;C in comparison to the RT condition (Figure 2), suggesting that temperature is important for protein conservation in the different AME obtained.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64460/64460fig02.jpg" /> 
Figure 2: Total protein concentration in AME affected by time and storage temperature. The concentration of protein extraction on AME was quantified before and after temperature and time storage conditions. Storage time evaluated was 6 days (black triangles), 12 days (pink triangles), 20 days (purple square), and 32 days (brown circles) in comparison with three different temperature conditions (RT &#176;C, 4 &#176;C, and -20 &#176;C). Basal protein concentration was similar among all AME. There was a significant difference in protein concentration in the AME of 20 and 32 days with respect to basal protein concentration at different temperature conditions. In each condition n = 3. Data is expressed as median of &#181;g/mL of protein &#177; SE *p &#60; 0.05 (S1 32 days vs. S1 20, 12, and 6 days at 4 &#176;C); (S1 32 days vs. S1 20, 12, and 6 days at -20 &#176;C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64460/64460fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Lumican concentration was affected by storage time and temperature conditions. Fewer concentration of lumican was found in the AME stored for 6, 20, and 32 days, compared with 12 days of storage. Significantly, the AME of 12 days had a higher concentration of lumican than 20 and 32 days of storage (p &#60; 0.05).
When lumican concentration in the AME was compared between storage temperatures, a higher concentration of lumican was found if stored at -20 &#176;C and 4 &#176;C for 12 days (Figure 3). Interestingly, even a higher (p &#60; 0.05) concentration of lumican was found in the 12 days AME if stored at -20 &#176;C in comparison with 4 &#176;C.
This suggests that the concentration of lumican is affected by temperature conditions and storage time, suggesting that the appropriate storage time and temperature to achieve the highest concentration of lumican is 12 days at -20 &#176;C.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64460/64460fig03.jpg" /> 
Figure 3: Total lumican concentration in AME affected by time and storage temperature. Lumican concentration was affected by storage time and temperature conditions. The concentration of lumican in AME was quantified before and after temperature and time storage conditions. Storage time evaluated was 6 days (black triangles), 12 days (pink triangles), 20 days (purple square), and 32 days (brown circles) in comparison with three different temperature conditions (RT &#176;C, 4 &#176;C, and -20 &#176;C). Lumican in the AME of 12 days was significantly higher in comparison with the AME of 32, 20, and 6 days, at temperature conditions of -20 &#176;C and 4 &#176;C. *p &#60; 0.05 (S1 12 days vs. S1 32, 20, and 6 days at 4 &#176;C); ***p &#60; 0.001 (S1 12 days vs. S1 32, 20 and 6 days at -20&#176;C). The highest concentration of lumican in AME was at 12 days stored at -20 &#176;C. ++p &#60; 0.01 (S1 12 days 4 &#176;C vs. S1 12 days -20 &#176;C). There was no significant difference between lumican in the AME of 32 and 20 days compared with storage temperature conditions (n.s). In each condition (n = 3), data are expressed as the median of ng/mL of protein. Data were normalized with respect to mg of tissue &#177; SE. (n.s.) not statistical significance. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64460/64460fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature at JoVE.com

Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature

The present protocol describes the extraction of lumican from the amniotic membrane (AM) and their storing conditions as AM extract (AME) at -20 &#176;C, 4 &#176;C, and room temperature (RT) for 6, 12, 20, and 32 days to quantify its proteins and lumican concentration.

Lumican is a small leucine-rich proteoglycan in the human amniotic membrane (AM) that promotes corneal epithelialization and the organization of collagen ...

lumican-extraction-from-amniotic-membrane-determination-its-storage

Nanyang Technological University

Research

JoVE Journal

Medicine

1.3K Views.  Instituto de Oftalmologia Fundacion Conde de Valenciana IAP. The present protocol describes the extraction of lumican from the amniotic membrane (AM) and their storing conditions as AM extract (AME) at -20 °C, 4 °C, and room temperature (RT) for 6, 12, 20, and 32 days to quantify its proteins and lumican concentration.

Video: Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Using an Ingestible Telemetric Temperature Pill to Assess Gastrointestinal Temperature During Exercise

Exercise results in an increase in core body temperature (Tc), which may reduce exercise performance and eventually can lead to the development of heat-related disorders. Therefore, accurate measurement of Tc during exercise is of great importance, especially in athletes who have to perform in challenging ambient conditions. In the current literature a number of methods have been described to measure the Tc (esophageal, external tympanic membrane, mouth or rectum). However, these methods are suboptimal to measure Tc during exercise since they are invasive, have a slow response or are influenced by environmental conditions. Studies described the use of an ingestible telemetric temperature pill as a reliable and valid method to assess gastrointestinal temperature (Tgi), which is a representative measurement of Tc. Therefore, the goal of this study was to provide a detailed description of the measurement of Tgi using an ingestible telemetric temperature pill. This study addresses important methodological factors that must be taken into account for an accurate measurement. It is recommended to read the instructions carefully in order to ensure that the ingestible telemetric temperature pill is a reliable method to assess Tgi at rest and during exercise.

This study describes an accurate, reliable and non-invasive technique to continuously measure gastrointestinal temperature during exercise. The ingestible telemetric temperature pill is suitable to measure gastrointestinal temperature in laboratory settings as well as in field based settings.

Exercise results in an increase in core body temperature (Tc), which may reduce exercise performance and eventually can lead to the development of ...

Esophageal Heat Transfer for Patient Temperature Control and Targeted Temperature Management

Controlling patient temperature is important for a wide variety of clinical conditions. Cooling to normal or below normal body temperature is often performed for neuroprotection after ischemic insult (e.g. hemorrhagic stroke, subarachnoid hemorrhage, cardiac arrest, or other hypoxic injury). Cooling from febrile states treats fever and reduces the negative effects of hyperthermia on injured neurons. Patients are warmed in the operating room to prevent inadvertent perioperative hypothermia, which is known to cause increased blood loss, wound infections, and myocardial injury, while also prolonging recovery time. There are many reported approaches for temperature management, including improvised methods that repurpose standard supplies (e.g., ice, chilled saline, fans, blankets) but more sophisticated technologies designed for temperature management are typically more successful in delivering an optimized protocol. Over the last decade, advanced technologies have developed around two heat transfer methods: surface devices (water blankets, forced-air warmers) or intravascular devices (sterile catheters requiring vascular placement). Recently, a novel device became available that is placed in the esophagus, analogous to a standard orogastric tube, that provides efficient heat transfer through the patient&#39;s core. The device connects to existing heat exchange units to allow automatic patient temperature management via a servo mechanism, using patient temperature from standard temperature sensors (rectal, Foley, or other core temperature sensors) as the input variable. This approach eliminates vascular placement complications (deep venous thrombosis, central line associated bloodstream infection), reduces obstruction to patient access, and causes less shivering when compared to surface approaches. Published data have also shown a high degree of accuracy and maintenance of target temperature using the esophageal approach to temperature management. Therefore, the purpose of this method is to provide a low-risk alternative method for controlling patient temperature in critical care settings.

This study presents a novel method to provide efficient patient temperature control for cooling or warming patients. A single use, triple lumen device is placed into the esophagus, analogous to a standard orogastric tube, and connects to existing heat exchange units to perform automatic patient temperature management.

Controlling patient temperature is important for a wide variety of clinical conditions. Cooling to normal or below normal body temperature is often ...

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) function are monitored by the change in voltage across the nasal epithelium after the superfusion of solutions that modify ion channel activity. This is enabled by the measurement of the potential difference between the subcutaneous compartment and the airway epithelium in the nostril, utilizing a catheter in contact with the inferior nasal turbinate.
The test allows the measurement of the stable baseline voltage and the successive net voltage changes after perfusion of 100 &#181;M amiloride, an inhibitor of Na+ reabsorption in Ringer&#39;s solution; a chloride-free solution containing amiloride to drive chloride secretion and 10 &#181;M isoproterenol in a chloride-free solution with amiloride to stimulate the cyclic adenosine monophosphate (cAMP)-dependent chloride conductance related to CFTR.
This technique has the advantage of demonstrating the electrophysiological properties of two key components establishing the hydration of the airway surface liquid of the respiratory epithelium, ENaC, and CFTR. Therefore, it is a useful research tool for phase 2 and proof of concept trials of agents that target CFTR and ENaC activity for the treatment of cystic fibrosis (CF) lung disease. It is also a key follow-up procedure to establish CFTR dysfunction when genetic testing and sweat testing are equivocal. Unlike sweat chloride, the test is relatively more time consuming and costly. It also requires operator training and expertise to conduct the test effectively. Inter- and intra-subject variability has been reported in this technique especially in young or uncooperative subjects. To assist with this concern, interpretation has been improved through a recently validated algorithm.

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD

Here, we present a standardized protocol to measure the nasal potential difference (NPD). Cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) function are evaluated by the change in the voltage across the nasal epithelium after superfusion of solutions that modify ion channel activity, providing an outcome measure.

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) ...

Proton Therapy Delivery and Its Clinical Application in Select Solid Tumor Malignancies

Radiation therapy is a frequently used modality for the treatment of solid cancers. Although the mechanisms of cell kill are similar for all forms of radiation, the in vivo properties of photon and proton beams differ greatly and maybe exploited to optimize clinical outcomes. In particular, proton particles lose energy in a predictable manner as they pass through the body. This property is used clinically to control the depth at which the proton beam is terminated, and to limit radiation dose beyond the target region. This strategy can allow for substantial reductions in radiation dose to normal tissues located just beyond a tumor target. However, the degradation of proton energy in the body remains highly sensitive to tissue density. As a consequence, any changes in tissue density during the course of treatment may significantly alter proton dosimetry. Such changes may occur through alterations in body weight, respiration, or bowel filling/gas, and may result in unfavorable dose deposition. In this manuscript, we provide a detailed method for the delivery of proton therapy using both passive scatter and pencil beam scanning techniques for prostate cancer. Although the described procedure directly pertains to prostate cancer patients, the method may be adapted and applied for the treatment of virtually all solid tumors. Our aim is to equip readers with a better understanding of proton therapy delivery and outcomes in order to facilitate the appropriate integration of this modality during cancer therapy.

The fundamentals of radiation planning and delivery for proton therapy using prostate cancer as a model are presented. The application of these principles to other selected disease sites highlights how proton radiotherapy may enhance clinical outcomes for cancer patients.

Radiation therapy is a frequently used modality for the treatment of solid cancers. Although the mechanisms of cell kill are similar for all forms of ...

Microelectrode Impalement Method to Record Membrane Potential from a Cannulated Middle Cerebral Artery

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ion channels and electrogenic pumps that regulate Vm in disease conditions could potentially alter Vm, vascular tone, and blood flow. Thus, a basic understanding of electrophysiology and the methods necessary to accurately record Vm in healthy and diseased states are essential. This method will allow modulating Vm using different pharmacological agents to restore Vm. Although there are several methods, each with its advantages and disadvantages, this article provides protocols to record Vm from cannulated resistance vessels such as the middle cerebral artery using the microelectrode impalement method. Middle cerebral arteries are allowed to gain myogenic tone in a myograph chamber, and the vessel wall is impaled using high resistance microelectrodes. The Vm signal is collected through an electrometer, digitized, and analyzed. This method provides an accurate reading of the Vm of a vessel wall without damaging the cells and without changing the membrane resistance.

The primary goal of this article is to provide details of how to record membrane potential (Vm) from the middle cerebral artery using the microelectrode impalement method. The cannulated middle cerebral artery is equilibrated to gain myogenic tone, and the vessel wall is impaled using high resistance microelectrodes.

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ...

Optimized Griess Reaction for UV-Vis and Naked-eye Determination of Anti-malarial Primaquine

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening infections caused by P. vivax and ovale. However, PMQ has unwanted adverse effects that lead&#160;to acute hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. There is a need to develop simple and reliable methods for PMQ determination with the purpose of dosage monitoring. In early 2019, we have reported an UV-Vis and naked-eye based approach for PMQ colorimetric quantification. The detection was based on a Griess-like reaction between PMQ and anilines, which can generate colored azo products. The detection limit for direct measurement of PMQ in synthetic urine is in the nanomolar range. Moreover, this method has shown great potential for PMQ quantification from human serum samples at clinically relevant concentrations. In this protocol, we will describe the technical details regarding the syntheses and characterization of colored azo products, the reagent preparation, and the procedures for PMQ determination.

This protocol describes a novel colorimetric method for antimalarial primaquine (PMQ) detection in synthetic urines and human serums.

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening ...