Scratch Assay Device for Wound Healing Studies

Transcription

1 Scratch Assay Device for Wound Healing Studies By Team Cell Scratcher: Hannah Morgan, Charles Merchant, and Zachariah Cribbin 1

2 Quick Start Guide 2

3 Comments: _ Table of Contents Cover Page Quick Start Guide Table of Contents Materials List Step-by-Step Instructions Assembling the Device Operating the Device Maintenance/Cleaning Needle Replacement Z-axis Replacement Scratcher Unit Cleaning/Sanitization of the Needle Cleaning/Sanitization of the Whole Device Glossary of Technical Terms General Terms 19 3

4 6.2 Definitions of device components Troubleshooting Z-axis piece Needle Scratcher unit..23 Materials List A. Top Sliding Piece (1) B. Medium rod (4) C. Top corner piece (4) D. Long rod (4) E. Steel base plate (1) F. Bottom corner piece (4) G. Short rod (4) 4

5 H. Z-axis holder (1) I. Reli-On tester (1) J. Needle Holder(1) K. Needle (1) Not Pictured L. Butterfly Knob (1) M. Nut (1) Comments: _ Step-by-Step Instructions Assembling the Device: Step 1: Center the top sliding piece on a flat surface in the figure shown to the right. Step 2: Insert the first of four medium-length rods into the top sliding piece. The rod is fully inserted when the score line on the rod lines up with the edge of the top sliding piece. 5

6 Step 3: Repeat the previous step with the three remaining medium length rods. Step 4: Insert top corner piece in the top right corner of the frame. Make sure vertical rod hole facing downward toward the floor. Step 5: Insert two of the four longest rods into the two remaining holes on the top corner piece. 6

7 Step 6- Insert the upper left and bottom right top corner pieces. Note: The joints may need to be loosened to insert the rods into the correct hole. Step 7: Before adding the last top corner piece, insert the two longest remaining rods into the corner piece as shown below. 7

8 Step 8: Insert the bottom left top corner piece into the assembly. Joints may need to be loosened first to correctly align all rods with the correct hole. The end result should look as shown below. Step 9: Set aside the top assembly and begin the bottom assembly with the steel base plate. 8

9 Step 10: Attach all four bottom corner pieces to each corner of the steel base plate. Step 11: Fully insert each of the four shortest rods into the bottom corner pieces on the steel base plate. 9

10 Step 12: Attach the top assembly onto the bottom assembly aligning the rods of the bottom assembly with the four remaining vertical holes of the top assembly. Step 13: Insert the z-axis holder into the top sliding piece and secure in place by tightening the butterfly knob on the top sliding piece. The frame of the device is now complete. Step 14: Place the Reli-On TM device in the Z-axis part as shown, with the grey button lined up with the hole in the device. 10

11 Step 15: Insert the needle into the needle holder as shown. 11

12 Step 16: Insert the needle holder into the end of the Reli-On TM device as shown. The device is now ready to be operated. 12

13 Comments: _ Operating the Device: Step 1: Place the well plate or Petri dish into the correct holder. Step 2: Pull back the plunger on the Reli-On TM device to load the needle. Step 3: Position the well plate holder on the magnetic platform so the needle of the scratcher is lined up with the well plate. Step 4: Loosen the butterfly knob and slide the Z-axis component down until the needle makes contact with the cells. Tighten the knob to lock the Z-axis component into place. Step 5: Press the grey button on the Reli-On TM device to propel the needle forward, creating a scratch in the cell monolayer. Step 6: Loosen the butterfly knob and move the Z-axis piece up, which will remove the needle from the well plate. Step 7: Sterilize the needle by dipping it into 70% ethanol, and allow it to evaporate. Step 8: Repeat steps 2-7 to perform another scratch. 13

14 Comments: _ Maintenance/Cleaning: Needle Replacement: The needle should only be used for about 50 scratches, therefore it is recommended that after 50 scratches the needle is replaced. 1. Remove needle holder from the Reli-On TM device. 2. Remove the used needle from the needle holder. 3. Place the new needle in the needle holder. 4. Reinsert the needle holder into the device. a. Remember that a calibration step must be done on the needle to ensure that accuracy of the device is maintained. 14

15 Z-axis Replacement: In the event that the z-axis piece is broken/ damaged it is recommended that it be replaced immediately in order to prevent skewed results. 1. Unscrew the butterfly knob to loosen the damaged z-axis piece from the top sliding piece. 2. Remove the scratcher unit from the damaged z-axis piece and then remove the z-axis piece from the top sliding piece. 3. Insert the new z-axis piece into the top sliding piece, and align the pieces, making sure that the tracks in the piece are able to easily move up and down. a. In the event that they do not allow free motion, make sure that the piece was properly installed. If it was not, remove and repeat step 3 from the beginning. 4. Screw the butterfly knob to lock the z-axis piece at the desired height. 15

16 Scratcher Unit Replacement: In the event that scratcher unit is damaged or no longer is generating a force to push the needle, it is recommended that the scratcher unit be replaced immediately. 1. Remove the needle holder from the scratcher unit (Reli-On TM ) device. 2. Remove the damaged scratcher unit from the z-axis movement piece. 3. Install the new scratcher unit: a. Place the new scratcher unit into the opening in the z-axis piece. b. Make sure that the push button is aligned with the opening on the z-axis piece, it should click into place. c. Make sure that the piece is secured within the z-axis piece, by gently moving it about to see if it will be easily removed 4. Reattach the needle holder to the new scratcher unit 5. Perform a scratch test without a sample to ensure that the scratcher unit is operating correctly. If device does not operate correctly redo from step 1. 16

17 Cleaning/Sanitization of the Needle: To sanitization the needle it is recommended that after each scratch the needle is dipped into 70% ethanol. 1. Adjust the height of the device using the z-axis piece. 2. Bring over a container of 70% ethanol and dip the visible parts of the needle into the ethanol. 3. Wait approximately 10 seconds to ensure that all the ethanol has evaporated. 4. Continue the procedure required to make a scratch in the further samples. WARNING: Do not attempt to sanitize the device using a flame technique. The 3D printed components will not withstand the heat a flame gives off, and they will melt. 17

18 Cleaning/Sanitization of the Whole Device: Recommended to clean the device after sample spills on the device or at least once a month to prevent contamination on the device. 1. Place the whole device in a separated area then spray down the device with a solution of 70% ethanol. 2. Do not saturate the device, just make sure that the whole device is lightly covered in the ethanol mist. 3. Wipe down the excess ethanol on the device and then allow the ethanol to evaporate from the device before the next use. WARNING: Do not put the device into an autoclave as can result in damage to device. The 3D printed components will not be able to withstand the high heat and pressure. 18

19 Comments: Glossary of Technical Terms: General Terms: In vitro scratch assay: An in vitro scratch assay is a method used to study cell migration patterns. The basic method is to create a wound in a cell monolayer by scratching through the cells. Upon scratching, the cells will migrate to close the gap. Definitions of Device Components: Needle: The needle is the component that makes contact with the cells and creates the scratch through the cell monolayer. Needle holder: The needle holder is the component where the needles are secured that interfaces with the spring loaded scratch mechanism. 19

20 Z-axis piece: Component of frame that houses the scratch mechanism and allows for height adjustment of scratcher. Scratcher Unit (Reli-On TM Device): Component that produces produces the spring force needed to move the needles. 20

21 Frame: The structure comprised of 3D printed parts and metal rods that provides structural stability for the scratcher. Platform: A steel base plate that doubles as structural support for the frame as well as a flat work surface to put well-plate holders on. Well Plate Holder(s): Plastic holders with magnetic bottoms prevent well-plates from moving as a scratch assay is performed. 21

22 Top Sliding Piece: Central component of frame where z-axis piece sits in. Comments: 22

23 Troubleshooting Z-axis piece: In the event that the Z-axis piece becomes damaged it is recommended that you contact a supervisor or request a new part. If current part is available see installation of Z-axis piece under maintenance/cleaning for instructions on page 15. Needle: In the event that the needle becomes damaged it is recommended that the needle be replaced immediately. Follow the procedure to replace needle, located on page 14. Scratcher unit: In the event that the scratcher unit malfunctions make sure that it is not jammed, if this does not fixed the issue it is possible that it is damaged. Therefore, go to the maintenance section and follow the procedure for scratcher unit replacement, located on page 16. Comments: _ 23