Zuivering van Biotinylated Cell Surface Proteins uit Rhipicephalus microplus Epitheliale darmcellen

Purification of biotinylated cell surface protein from Rhipicephalus microplus epithelial gut cells. Within this study, a modified Percoll gradient-based methodology was utilized to isolate individual epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for the utilization in downstream applications.

Dissection of the gut epithelium from semi-engorged Rhipicephalus microplus. You will need the following materials. Adhere a strip of duct tape to the bottom of a Petri dish.

Add a drop of Super Glue to the tape. Place the tick ventral side down on the Super Glue, allowing it to dry. Completely submerge the tick in 100 mL of PBS.

Utilizing a size 11 scalpel, cut from the top of the eyes of to the bottom festoons on both sides of the tick. Using sterile forceps, completely remove the scutum and alloscutum to expose the internal organs. Remove the fine white threadlike organs and other membranes.

Remove the gut using forceps. Store gut in ice-cold HBSS and PIC. Epithelial cell disassociation.

You will need the following materials. Pour the dissected gut onto a 70 micron cell strainer inside a 50 mL Falcon tube. As a vacuum may form within the Falcon tube, lift the filter to allow for air circulation.

Flush the gut tissue with 50 mL ice-cold HBSS with PIC. Resuspend guts in 30 mL ice-cold HBSS with PIC. If necessary, use a sterile scraper to assist in gut resuspension.

Centrifuge at 500 g. Remove the supernatant and repeat the wash process three times. Resuspend the gut cells in 10 mL of DMEM, 2%FCS, 5 millimolar EDTA, one millimolar TCEP and PIC.

Mix gently. Incubate for 60 minutes at 37 degrees Celsius under slow rotation using a roller. Filter the suspension through a 250 micron cell strainer.

Vortex the flow through. And filter through a 70 micron cell strainer, collecting the remaining flow through. Centrifuge the suspension.

Isolation of single epithelial cells using a Percoll gradient. You will need the following materials. By filtering through AP15 pre-filter paper, prepare 40%and 20%Percoll in Milli-Q water.

Load a sterile syringe with the Percoll solution. Apply it to the filter rig and slowly discharge the Percoll. Cool at four degrees Celsius for an hour prior to layering the gradient.

Set up the peristaltic pump as shown. Using a peristaltic pump set at the lowest speed, layer three mL of 40%Percoll into a 16 mL ultracentrifuge tube. Allow it to settle on ice for 15 minutes.

Speed of the pump should lead to a less than one mL per minute flow rate. Tilting the tube to a 45 degree angle, use the peristaltic pump to layer the 20%Percoll on top of the 40%layer. Ensure that the output tube is resting against the centrifuge tube to prevent droplets mixing with lower layers.

Speed of the pump should lead to less than one mL per minute flow rate. Allow the layers to settle on ice for 15 minutes. Using the peristaltic pump at less than one mL per minute flow rate to layer three mL of DMEM solution containing the isolated epithelial cells over the 20 to 40%Percoll gradient.

Ensure the output tube is resting against the centrifuge tube to prevent droplets mixing with the lower layers, disrupting the gradient. Centrifuge at 600 g for 10 minutes. Collect interphases between the DMEM, 20%Percoll gradient, and the 20%40%Percoll gradients to isolate epithelial single cells.

Cell surface protein biotinylation and the isolation of biotinylated surface proteins. You will need the following materials. Biotinylate 100 microliters of single-cell epithelial cells using Biotin Type A Conjugation Kit as per manufacturer's instructions.

Add 100 microliters of PBS, 1%Triton X-100, 10%glycerol, 100 micromolar oxidized glutathione, and PIC to the biotinylated cells. Incubate on ice for an hour with gentle mixing every 10 minutes. Centrifuge the cell extract at 20, 000 g at four degrees Celsius for 20 minutes and set aside.

Prepare 50 microliters of streptavidin magnetic beads by washing with 1, 000 microliters of TBS 1%Tween-20. Mix gently by flicking with your finger. Place the tube into a magnetic stand, collecting the beads against the side of the tube.

Remove and discard the supernatant. Combine 300 microliters of biotinylated cell surface proteins with washed magnetic beads. Incubate for two hours at room temperature with agitation.

Collect the beads with the magnetic stand, remove, and discard the supernatant. Add 300 microliters of TBS 1%Tween-20 to the tube, gently mixing to resuspend the beads. Collect beads, remove, and discard the supernatant.

Repeat this wash step twice. Add 100 microliters of the 1 molar glycine pH two to the magnetic beads. And incubate at room temperature for five minutes.

Collect beads and remove supernatant containing eluted biotinylated surface proteins. Figure one is a representation of the protocol utilized to isolate epithelial cells and their surface proteins from a whole tick. Utilizing this protocol, approximately 1.2 by 10 to the seven cells per mL with 75 to 80%viability and 20 to 24 micrograms of purified biotinylated surface proteins can be purified from an initial dissection of 50 ticks.

Representative fluorescence microscopy imagery of tick get epithelial cells prepared using this protocol are shown in figure 2A and 2B. Cells appear as singular, spherical, smooth surface morphology and a consistent size throughout the sample. Poor isolations are visualized to contain incomplete dissociation of individual epithelial cells with varying cell populations identified through the varying cell sizes and morphologies.

Cross-contamination from host proteins, figure 3A, can be minimized by adequately rinsing tick guts to produce a white or clear Percoll gradient, figure 3B. Comparison of proteins by SDS-PAGE, figure 4A, silver stain, figure 4B, dot blot, figure five, and ELISA, figure six, indicates that the described methodologies successfully purified biotinylated surface proteins from individual epithelial cells isolated from Rhipicephalus microplus tick guts. In conclusion, the methods utilized in this study successfully isolated individual epithelial cells from the whole gut of Rhipicephalus microplus.

Proteins from the surface of Rhipicephalus microplus epithelial cells were obtained for further analysis and studies. Finally, the protocol developed has demonstrated the potential yield of individual epithelial cells from the tick gut and the efficiency of biotinylated surface proteins of the cell. The protocol developed can be utilized in any tick species of economic significance in an effort to investigate tick-host interactions by studying the membrane protein composition of the gut.