John Herich

  1. 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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  2. Mixing in Small HPLC Vials

    V&P Scientific makes miniature magnetic stir discs which are encapsulated in PVDF or PEEK. These magnetic discs are well suited for stirring inside small HPLC vials.

    Thomson SINGLE StEP™ and eXtractor 3D™ filter Vials are designed to speed up sample prep and analysis. The plunger filter with different membranes nestles into the vial while simultaneously filtering and readying the sample for any autosampler. This is a process that minimizes any loss of sample eliminating multiple transfers. Thomson filter vials are compatible with most standard autosamplers; such as Agilent® and Waters® including the UHPLC™.

    This photo shows a Thomson eXtractor 3D™ filter vial next to a couple of V&P PVDF encapsulated magnetic stir discs.

    These stir discs have optimal liquid mixing performance with our VP 710D2 Multi Stirrus™.  This design is well suited for mixing different solutions or dissolution of powders.

    When used in conjunction with the VP 710D2 Multi Stirrus™, more than 100 HPLC vials can be mixed simultaneously.  If resolubilizing your compounds after refrigeration, or aiding in the initial solubilization of compounds is important for your procedure, then this is an easy solution for you.

     This video demonstrates stirring in both chambers, and stirring in a single chamber after the filtration step has been completed.

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  3. Stop Losing Precious Reagents to Dead-Volume!

    The VP 534-ALL is a low profile 384 reagent reservoir made with hydrophilic coated aluminum. The hydrophilic coating significantly reduces the dead-volume lost because of surface tension effects of the reservoir. The reservoir is designed to be washed and reused, and can be autoclaved for sterility.

    V-Groove Low Dead-Volume Reservoir:
    This reagent reservoir conforms to SBS standard dimensions: 127.7 mm long / 85.5 mm wide / 13.5 mm deep cavity / 15 mm tall.

    The standard microplate dimensions enable the reservoir to be used with robotic plate handlers or stackers. The specially designed hydrophilic coated grooves in the reservoir bottom reduce reagent waste, and allow consistent aspiration of minimal amounts of reagent. Dead volumes below 3 ml are possible depending on the solution, and aliquot volume.
         The hydrophilic surface allows liquids to spread out evenly, requiring only a minimal amount of starting reagent to cover the bottom. Starting volumes as low as 4.5 ml will spread, and allow 384 channel based aspiration.

    This is a photo of the reservoir with 5 ml of RPMI 1640 tissue culture medium:



    These are photos that compare 5ml media in various reservoirs.
    VP 534-ALL {lower left} shows superior distribution:
    The photo below shows 5ul droplets dispensed from a liquid handling robot equipped with a 384 channel pipet head; the droplets were dispensed onto a microplate lid. The robot aspirated 1.96ml media from the VP 534-ALL reservoir which had been filled with only 4.5ml of starting material. The droplets were consistent in size and the overall weight of the plate before and after dispensing indicated that ~5.1ul was delivered to each spot:
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