immunostaining of cells encapsulated in a 3d hydrogel system

Immunostaining of Cells Encapsulated in a 3D Hydrogel System

First, aspirate the medium from each well. Then, wash the wells with 1 milliliter of warm DPBS. After washing is completed, add 750 microliters of fixation solution to each well. Then, incubate the 24-well plate at 37 degrees Celsius for 30 minutes. After the incubation, aspirate the fixation solution from each of the wells, and discard the fixative in a hazardous waste container. Next, add 1 milliliter of DPBS to the sample and immediately discard the DPBS into a hazardous waste container.
Next, add 750 microliters of permeabilization solution to permeabilize the sample for one hour at room temperature on the rocker, at 15 revolutions per minute. After one hour, aspirate the permeabilization solution, and add 750 microliters of blocking solution to the sample. Leave the sample on the rocker at room temperature for three hours. Then, dilute the desired primary antibodies with the antibody dilution solution.
Next, add 500 microliters of the antibody solution to each sample. Incubate the samples overnight at 4 degrees Celsius. The following day, aspirate the primary antibody solution. Then, wash the samples with PBST for one hour thrice, at room temperature, at 15 revolutions per minute.
Next, dilute the appropriate secondary antibodies and 5 mg/ml of DAPI stock solution to achieve a final concentration of 2.5 micrograms per milliliter. Then, add 500 microliters of the solution to each sample. Next, use aluminum foil to cover the 24-well plate and incubate the plate at 4 degrees Celsius overnight.
The next day, aspirate the secondary antibody solution and wash the samples with PBST thrice, each for 30 minutes at room temperature on a rocker. Next, position a drop of hard set mounting medium on the surface of a glass slide and place the sample on the mounting medium. Finally, use nail polish to seal the samples to the glass cover slide to prevent contamination or sample movement.

immunostaining-of-cells-encapsulated-in-a-3d-hydrogel-system

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1. Cell Encapsulation in 3D Elastin-like Protein (ELP) Hydrogels
<ol>
	<li>Preparation of silicone molds
	<ol>
		<li>Use a biopsy punch with the desired diameter to create holes in a 0.5 mm thick silicone sheet and cut out a square around each hole. Repeat for the number of molds desired. 
		&#8203;NOTE: The diameter and thickness of the molds can be adjusted for the particular application and cell culture conditions. In practice, for cell cultures of 50 x 106 cells/mL, 0.5 mm thick molds with 4 mm and 5 mm diameters are recommended for sufficient DNA/RNA and protein extraction, respectively. For immunostaining, a 2 mm biopsy punch can be used to instead create three adjacent holes per square mold so that three replicates can be stained per well.</li>
		<li>Remove the plastic wrap on each side of the individual mold with tweezers. 
		NOTE: Avoid contact with the exposed silicone surface as contamination can decrease future plasma bonding efficiency.</li>
		<li>Using tweezers, arrange the same number of bare silicone molds and glass coverslips (No. 1, 12 mm diameter) in alternating rows on the inverted lid of a 48-well plate. Once completed, cover the lid of the 48-well plate to avoid contamination.</li>
		<li>Oxygen plasma treat the entire 48-well plate lid (i.e., molds and glass slides). Immediately after, use forceps to invert the silicone mold onto the adjacent glass coverslip. Press firmly on the mold to ensure bonding. Let the molds incubate at room temperature for 1 h. 
		NOTE: The duration of oxygen plasma treatment will vary depending on the instrument used. Typical conditions for our instrument are an operating gas pressure window between 0.3-4 mbar, oxygen gas flow at 20 cm3/min, and sample exposure to plasma for 10-20 s.</li>
		<li>Sterilize the molds by autoclaving. Store the molds at room temperature in a sterile environment until use. 
		NOTE: The molds can be stored at this stage indefinitely.</li>
	</ol>
	</li>
	<li>Preparation of elastin-like protein stock solution&#8203;
	<ol>
		<li>Remove lyophilized ELP from 4 &#176;C storage. Warm the protein to room temperature before opening the tube to ensure no condensation builds on the protein over repeated use.</li>
		<li>Dissolve ELP in Dulbecco&#39;s phosphate-buffered saline (DPBS) at 4 &#176;C with constant agitation (i.e., spinning) overnight. 
		NOTE: ELP stock solution concentration will be further diluted with the addition of crosslinker solution at a user-defined volumetric ratio. For example, for a 3% (w/v) final ELP concentration, prepare a 3.75% (w/v) ELP stock solution that will be diluted at a 4:1 volumetric ratio of ELP solution:crosslinker solution. Adjust the concentration as necessary for desired application.</li>
		<li>Sterile filter ELP stock solution using a 0.22 &#181;m syringe filter. Store ELP on ice when not in use.</li>
	</ol>
	</li>
	<li>In the biosafety cabinet, transfer the sterile molds using sterile tweezers to a 24-well tissue-culture plate.</li>
	<li>Preparation of tetrakis(hydroxymethyl)phosphonium chloride (THPC) stock solution&#8203;
	<ol>
		<li>Dilute THPC in DPBS just prior to use. Adjust the concentration of THPC solution according to the final ELP concentration and desired crosslinking ratio. 
		NOTE: For the protocol, a final ELP concentration of 3% (w/v) will be made by mixing the ELP stock solution with the THPC solution at a volumetric ratio of 4:1. Adjust as necessary.</li>
		<li>Add 2.6 &#181;L of THPC solution (80% in water) to 997.4 &#181;L of DPBS. 
		NOTE: This concentration of THPC corresponds to a 1:1 stoichiometric ratio of hydroxy methyl groups on THPC and primary amines on the ELP protein when mixing solutions at the described 4:1 volumetric ratio for a final 3% (w/v) ELP hydrogel. The THPC solution is viscous and droplets of solution may stick to the side of the pipette tip. For accurate concentrations, avoid such droplets when diluting into DPBS. Purge the THPC stock container with nitrogen gas to prevent oxidation of the phosphine and inactivation of the crosslinker.</li>
		<li>Vortex the solution to mix and keep on ice.</li>
		<li>Sterile filter the THPC stock solution using a 0.22 &#181;m syringe filter. Dilute the THPC stock solution further with sterile DPBS to achieve lower stoichiometric crosslinking ratios (e.g., 0.5:1 or 0.75:1). 
		NOTE: THPC is oxygen-sensitive, and the diluted solution should be used within a few hours after preparation.</li>
	</ol>
	</li>
	<li>Dissociate the cells by incubation with Trypsin-EDTA into a single-cell suspension, pellet the cells, and count the cells re-suspended in medium using a hemocytometer. 
	NOTE: Exact protocols for this step will depend heavily on desired cell type and application. For the neural progenitor cells used throughout this protocol, a 0.025% Trypsin-EDTA incubation at room temperature for 1.5 min was conducted. The cells were pelleted at 200 x g for 2 min. For increased cell viability, cells should be suspended in normal medium conditions. Typical cell densities used for cell encapsulation range from 1 - 50 x 106 cells/mL of final hydrogel volume. Adjust as necessary.</li>
	<li>Aliquot the desired number of cells into a sterile 1.5 mL centrifuge tube.</li>
	<li>Centrifuge the cells at ~200 x g for 3 min. Carefully, aspirate the supernatant and keep the cell pellet on ice. 
	NOTE: It is critical to completely aspirate all the supernatant to mitigate the further dilution of the ELP and THPC crosslinker. Specific centrifugation speeds will vary with cell type.</li>
	<li>Re-suspend the cell pellet in the ELP stock solution such that the volume is 80% of the final volume (assuming a 4:1 ratio of ELP solution:THPC solution). Pipette mix 20-25 times to produce a homogenous mixture of the cells and ELP. 
	NOTE: Avoid gripping the bottom of the tube to mitigate temperature increase and subsequent phase transition of ELP. Each 2 mm mold with three replicates requires 7.5 &#181;L of final volume (i.e., 6 &#181;L of ELP stock solution and 1.5 &#181;L of THPC stock solution) divided equally into the 3 holes (i.e., 2.5 &#181;L final volume per hole). For the 4 and 5 mm molds, a final volume of 7.5 &#181;L and 15.5 &#181;L is required, respectively.</li>
	<li>Add THPC stock solution to the cell/ELP suspension for the remaining 20% final volume. Pipette mix 20-25 times to produce a homogenous mixture.</li>
	<li>Immediately pipette the corresponding final volumes of cell/ELP/THPC mixture into each mold by a circular motion. Repeat for all molds.</li>
	<li>Incubate the samples at room temperature for 15 min, followed by an additional incubation at 37 &#176;C for 15 min. 
	NOTE: The first incubation period will help facilitate initial crosslinking of the hydrogel before increasing the temperature to that above the ELP&#39;s lower critical solution temperature (LCST) and inducing its thermal phase segregation.</li>
	<li>Slowly add 750 &#181;L of warm cell culture medium to each well of the 24-well plate, avoiding disrupting the gels.</li>
	<li>Incubate the hydrogels at 37 &#176;C for 7 days. 
	NOTE: Full medium changes are recommended every 1-2 days depending on cell type. To limit the stress on the gel, use a glass Pasteur pipette affixed with a 200 &#181;L pipette tip when aspirating medium from the well.</li>
</ol>
2. Immunocytochemistry of Cells in 3D ELP Hydrogels
<ol>
	<li>Prepare the fixation solution by mixing 10 mL 16% (w/v) paraformaldehyde (PFA) in 30 of mL DPBS. Warm the solution to 37 &#176;C.</li>
	<li>Aspirate the medium from the 24-well plate and gently wash with 1 mL of DPBS.</li>
	<li>Add 750 &#181;L of the fixation solution to each well and incubate at 37 &#176;C for 30 min. Do not use the tissue culture incubator to avoid contamination of other cultures with PFA vapor.</li>
	<li>Carefully aspirate the fixation solution from each well, discarding the PFA to an appropriate hazardous waste container. 
	CAUTION: Exposure to PFA can cause skin and eye irritation. Wear gloves/safety glasses and work in a chemical fume hood.</li>
	<li>Add 1 mL of DPBS to each sample. Immediately aspirate the DPBS and discard into PFA waste container.</li>
	<li>Wash the samples twice with 1 mL of DPBS for 10 min each. 
	NOTE: Samples can be stored in DPBS at 4 &#176;C for up to a week after sealing the plate with Parafilm.</li>
	<li>Permeabilize each sample with 750 &#181;L of permeabilization solution (100 mL of DPBS and 0.25 mL of Triton X-100; PBST) for 1 h at room temperature on a rocker at 15 rpm.</li>
	<li>Aspirate the PBST and add 750 &#181;L of blocking solution (95 mL of PBS, 5 g of bovine serum albumin (BSA), 5 mL of serum, and 0.5 mL of Triton X-100) to each sample. Incubate at room temperature for 3 h on a rocker at 15 rpm. 
	NOTE: The blocking solution should contain serum from the host species in which the secondary antibodies were raised (e.g., goat, donkey) and sterile-filtered through a 0.22 &#181;m filter prior to use.</li>
	<li>Prepare the antibody dilution solution (97 mL of DPBS, 2.5 g of BSA, 2.5 mL of serum (from same host species from which EPL is prepared), and 0.5 mL of Triton X-100). Dilute the primary antibody using the antibody dilution solution and add 500 &#181;L of the solution to each sample. Seal the plate with Parafilm and incubate overnight at 4 &#176;C on a rocker. 
	NOTE: Nestin and Sox2 primary antibodies were diluted 1:400 in antibody dilution solution from the manufacturers&#39; original concentration.</li>
	<li>Aspirate the antibody solution from each sample and wash the samples with PBST for 60 min at room temperature on a rocker at 15 rpm. Repeat the wash step 3 times.</li>
	<li>Dilute the secondary antibodies and 5 mg/mL stock DAPI (1:2,000) using the antibody dilution solution and add 500 &#181;L of the solution to each sample. Cover the 24-well plate with aluminum foil and incubate overnight at 4 &#176;C on a rocker. 
	NOTE: As the secondary antibodies are light sensitive, the samples must be protected from photobleaching for all subsequent steps. Goat anti-mouse and goat anti-rabbit secondary antibodies were diluted 1:500 in antibody dilution solution from the manufacturers&#39; original concentration.</li>
	<li>Aspirate the antibody solution from each sample and wash the samples with PBST for 30 min at room temperature on the rocker. Repeat the wash step 3 times.</li>
	<li>Position a drop of hard-set mounting medium onto the surface of a glass slide. Using forceps, remove excess solution from the mold by lightly blotting the edge of the mold on a paper towel. Carefully place the mold upside down onto the mounting medium and avoid introducing bubbles.</li>
	<li>Allow the mounting medium to harden for 48 h at room temperature. Store the samples at room temperature or 4 &#176;C. Allow the mounting medium to fully set for 48 h before imaging as refractive index of the medium will change over the course of hardening. Seal the samples to the glass cover slide with clear nail polish to mitigate contamination or sample movement.</li>
	<li>Image the samples using a confocal microscope.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Encapsulation in 3D ELP Hydrogels</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m syringe filters</td>
			<td>Millipore</td>
			<td>SLGV004SL</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mm thick silicone sheet</td>
			<td>Electron Microscopy Science</td>
			<td>70338-05</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture plates</td>
			<td>Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Biopsy Punch (2 mm)</td>
			<td>Integra Miltex</td>
			<td>33-31</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Biopsy Punch (4 mm)</td>
			<td>Integra Miltex</td>
			<td>33-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Biopsy Punch (5 mm)</td>
			<td>Integra Miltex</td>
			<td>33-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate buffered saline (DPBS)</td>
			<td>Corning</td>
			<td>21-031-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 1 12 mm glass coverslips</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-545-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrakis(hydroxymethyl)phosphonium chloride (THPC)</td>
			<td>Sigma-Aldrich</td>
			<td>404861-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% Tryspin/EDTA</td>
			<td>Thermo Fisher</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunocytochemistry of Cells in 3D ELP Hydrogels</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>16% (w/v) Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15701</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Roche</td>
			<td>3116956001</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Molecular Probes</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Serum</td>
			<td>Lampire Biological Labs</td>
			<td>7332100</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse Secondary Antibody (AF488)</td>
			<td>Molecular Probes</td>
			<td>A-11017</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Secondary Antibody (AF546)</td>
			<td>Molecular Probes</td>
			<td>A-11071</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Serum</td>
			<td>Gibco</td>
			<td>16210-072</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Nestin Primary Antibody</td>
			<td>BD Pharmingen</td>
			<td>556309</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Sox2 Primary Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>23064S</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail Polish</td>
			<td>Electron Microscopy Sciences</td>
			<td>72180</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield Hardset Mounting Medium</td>
			<td>Vector Labs</td>
			<td>H-1400</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: LeSavage, B. L., et al. <a href="https://app.jove.com/v/57739">Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D.</a> J. Vis. Exp. (2018).
This video demonstrates a technique for immunostaining cells encapsulated in a 3D hydrogel. The hydrogel culture containing neural progenitor cells is fixed, permeabilized, and treated with a blocking solution to prevent non-specific immunostaining. A primary antibody cocktail is added to bind specific proteins, followed by fluorescently labeled secondary antibodies and a nuclear stain. The sample is then mounted on a slide and observed under a confocal microscope.

1. Cell Encapsulation in 3D Elastin-like Protein (ELP) Hydrogels

	Preparation of silicone molds
	
		Use a biopsy punch with the desired diameter to ...

Take a multi-well plate containing mammalian cells encapsulated in a hydrogel system mimicking the natural extracellular matrix environment.
Remove the culture medium. Treat with a fixation buffer to crosslink the proteins, preserving cellular integrity.
Add a solution containing detergent to permeabilize the cellular membrane.
Introduce a blocking solution to prevent non-specific antibody binding in subsequent steps.
Add a primary antibody cocktail that enters cells and binds specifically to cytoplasmic intermediate filaments and the target transcription factor.
Remove the unbound antibodies. Introduce fluorescently labeled secondary antibodies and a nuclear stain to label the DNA fluorescently.
Remove the unbound molecules.
Mount the hydrogel culture onto a slide using a mounting medium.
Use a confocal microscope to observe the immunostained cells.

37 vistas. Inmunotinción de células encapsuladas en un sistema de hidrogel 3D

Video: Inmunotinción de células encapsuladas en un sistema de hidrogel 3D - Experimento

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

Ácido hialurónico basado en hidrogeles de 3 dimensiones cultura de células de Glioblastoma derivados del paciente

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Investigación

JoVE Journal

Bioingeniería

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

Generación escalable de organoides cerebelosos maduros a partir de células madre pluripotentes humanas y caracterización por inmunosuización

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Identificación de marcadores de superficie celular de células madre neuronales primarias y células progenitoras por etiquetado metabólico de sialoglicano

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Immunostaining of Cells Encapsulated in a 3D Hydrogel System

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