Pin Tools

  1. Factors Affecting Pin Delivery Volumes in Robotic Applications

         The most important characteristics of pins, and the primary determinates of the volume of liquid transferred, are the physical features and the speed of withdrawal. The list below summarizes all the factors that contribute to the volume delivered. With each application these factors can be controlled and standardized so that the delivery volumes are very reproducible. With most applications the CV's of pin volume transfers are less than 5%.

    1. Pin Diameter. The greater the diameter the larger the hanging drop and the greater the volume transferred. We have developed very precise methods and treatments of the pins to achieve very reproducible volume transfers.

    2. Volume of slot in the pin. We use a very exact EDM process to create precision volume slots. The larger the slot the greater the volume transferred.

    3. Speed of removal of pin from source liquid. The faster the speed of pin removal from the liquid the greater the volume transferred as the liquid does not have time to drain from the sides of the pin. Increasing the speed of withdrawal from the source plate by 7- fold will increase the volume delivered by as much as 3 fold in a linear relationship. This phenomenon can be exploited to expand the range of delivery volume for a single pin. The chart below illustrates the effects of increasing the speed 7- fold. The speed range is from 0.78 cm/sec to 5.7 cm/sec.  Use this chart to select the pin that will deliver in your desired range.

    4. Depth to which the pin is submerged in source plate. The greater submersion depth the greater the volume transferred on the sides of the pin.

    5. Depth to which the pin is submerged in recipient plate liquid. The greater the depth the greater the volume transferred as long as it is at least equal to the depth of the source well.

    6. Microplate well diameter. The larger the well diameter the greater the volume transferred. This has to do with the proximity of the well meniscus affecting the surface tension of liquid in the middle of the well where the pin removes the liquid.  See this table comparing 96 and 384 well microplates and the volumes transferred.

    7. Surface tension of the pin. The greater the surface tension the smaller the volume transferred.  Hydrophobic/lipophobic coatings have the most effect on the larger diameter pins and at the lower speeds of withdrawal.

    8. Surface tension of the liquid being transferred. The greater the surface tension the smaller the volume transferred.

    9. Speed of pin striking recipient dry plate. The faster the striking speed the greater the volume transferred.

    10. Surface tension of the dry plate. The less the surface tension the greater the volume transferred.

    Select The Volume You Need To Transfer For Your Application

    Delivery Volume Ranges

    Delivery volume range is determined by speed of withdrawal from source liquid.

    slow speed  = 0.78 cm/sec = low volume delivery range

    fast speed  =  5.70 cm/sec = high volume delivery range

    Follow this link to assay methods

    The links below are to delivery volume range tables for uncoated and hydrophobic coated delivering either DMSO or Aqueous solutions in liquid to liquid transfers.

    DMSO TRANSFERS WITH UNCOATED AND HYDROPHOBIC COATED PINS

    AQUEOUS TRANSFERS WITH HYDROPHOBIC COATED PINS

    AQUEOUS TRANSFERS WITH UNCOATED STAINLESS STEEL PINS

    A Guide for Selecting the Right Pin for Your Application

    1. What volume do you need to transfer? DMSO? Aqueous? See these tables to determine your options. Also consider custom slot pins.

    2. What volume is in the source plate wells? If it is small a slot pin will have a definite advantage.

    3. Will the source plate have wells with significantly different levels of liquid? Cherry picked source plate or edge drying effect?. If yes and if the absolute volume transferred is critical, then select the largest slot pin that is in your transfer range. This will minimize the effect of liquid height on the volume of liquid carried on the sides of the pin. Also consider custom slot pins.

    4. Does the material transferred bind non-specifically to stainless steel? If yes then select the Hydrophobic or lipophobic coated pins. If no, select the uncoated pins.

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  2. Wound Healing Using 384 Well Microplates

    Introduction:

           Wound healing involves cell migration and invasion which are processes that offer rich targets for intervention in key physiologic and pathologic pathways. With the advent of high-throughput and high content imaging systems, there has been a movement towards the use of physiologically relevant, phenotypic cell-based assays earlier in the testing paradigm. This allows more effective identification of lead compounds and recognition of undesirable effects sooner in the drug discovery screening process. At V&P Scientific, we have created tools for mechanically scratching the cell substrate with a 384 pin array.  Scientists can create characteristically sized wounds in all wells of a 384 well plate.

     

    Rationale:

           Many of our customers have used our pin tools to "wound" cell culture monolayers and then study the effects of different treatments on wound healing. Initially, our standard 96 wound healing pin tools and a special wounding Library Copier (VP 381NW, VP 381NW4.5 or VP 381NW5) were used to make the wounds. For most applications, these wounding tools give good results. However, some customers who were using 384 well plates needed the wounds to be more precisely localized. For these customers, we have developed specialized pin tools that have very tight hole tolerances in the floating fixture so the wounds are consistently located in the same position in each well.

           A recent addition to our line of wounding pin tools are pins with a 0.05 mm layer of Parylene coating, deposited using a vapor deposition process to produce a soft lining on the pin tips. Each pin now acts as “eraser” to remove the cell monolayer without scratching the plastic well surface under the monolayer. Another new innovation and problem solved only by V&P Scientific.

           Another factor that can lead to wound variation is a loose fit between the microplate and the registration device (e.g., our VP 381NW Library Copier).  This is due to variation in the molds used to make the microplates. One way of dealing with the problem is to use our adjustable Library Copiers (VP 381NWGV4.5 or VP 381NWGH4.5).

     

    Results:

           We recently developed a new Library Copier for creating wounds in 384 well microplates in the horizontal direction (VP 381NWGH4.5). We added a second set of slots to this library copier which allows the user to use a 384 pin tool containing only 192, or 96 pins which significantly reduces the cost of the wound creating pin tool, and still allows for the production of 384 wounds by simply changing the pin tool and library copier orientation.

     

    These pictures show the V&P fixture, AFIX384FPWP, filled with 192 or 96 FP-WP pins:

     

    The following pictures show the V&P fixture, AFIX384FPWP, filled with 192 or 96 FP-WP pins, being used to produce wounds in 384 plates half-a-plate at a time, or in quadrants by using the library copier: VP 381NWGH4.5.  Standard carbon paper was used to trace the scratch produced by the wound healing tool.

     

     

    Top Half of 384 Well Plate

     

    Bottom Half of 384 Well Plate

    Result, a Complete 384 Well Plate

     

    1st Quadrant(Upper Left) of a 384 Well Plate

     

    2nd Quadrant(Lower Left) of a 384 Well Plate

     

    Rotate Plate 180 Degrees, Repeat Scratches, Result a complete 384 Well Plate:

     

    CHO-K1 (ATCC® CCL-61™) Cells Before and After Scratching:

     Before                                                                                                                                                                                                  After

     

    Summary:

     

           V&P Scientific's pin tools are an effective way to perform wound healing experiments. The use of 384 well plates allows for high-throughput wound creation allowing many compounds to be screened for effects on cell migration or invasion. The use of V&P Scientific's Library Copiers helps to create wounds uniform in length and width. Parylene coated pins produce a soft lining on the pin tips. Each pin now acts as an “eraser” to remove the cell monolayer without scratching the plastic well surface under the monolayer.

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  3. Cleaning V&P Pins with Caustic Soda

         For daily and between-transfer cleaning, V&P Scientific recommends the VP 110 Pin Cleaning Solution. This proprietary liquid uses a combination of acid and detergent to remove soiling caused by cell debris, salts, proteins and nucleic acids. However, it may become necessary to use a more aggressive cleaning agent; caked on protein, organic soils, or burnt-on carbon deposits caused by flaming handheld replicators can affect transfer volume and consistency, and can be difficult to remove.

         For these cases, a solution of Sodium Hydroxide, or caustic soda, is recommended for deep cleaning. V&P Scientific pins are made from stainless steel and are not damaged by high pH, though, we recommend against exceeding solutions of more than 3% sodium hydroxide to avoid pitting of the stainless steel. Start with a 1% solution and increase for subsequent cleanings if needed. For best results, use the NaOH solution hot- between 60 and 80 degrees C. You can adjust the concentration, temperature, and residence time of the pins in the solution to accommodate the level of soiling.

         A sonicator may also be used to aid in cleaning, and can help maintain the temperature of the cleaning solution. Be sure to cover the pin tool during sonication to avoid splashing and wear all appropriate PPE as hot caustic solutions are extremely dangerous. An example cleaning procedure is described below:

    1.      Prepare a solution of 2% NaOH in distilled or deionized water.

    2.      Heat the solution to 60C.

    3.      Immerse the Pin Tool in the hot solution, making sure that the float plate and frames do not come    into contact with the solution. Only the pins should be submerged.

    4.      Sonicate with heat on 60C for 30 minutes.

    5.      Remove pins from cleaning solution and blot using V&P Scientific's VP 540DB blotting station.

    6.      Rinse pins well with distilled water. If any cleaning solution has splashed onto float plates, rinse the float plates. Blot.

    7.      Rinse pins well with 70% ethanol. If the float plates are wet, rinse the float plates with ethanol. Blot.

    8.      Air Dry Pin Tool

    9.      If pins are fully clean, perform the standard cleaning procedure with VP 110 Pin Cleaning solution and return pin tool to service.

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