Adaptive Laboratory Evolution (ALE) is a frequently used method in biology to study molecular evolution and adaptive changes in microbial populations over a long term period of time and under specific growing conditions. With the recent breakthroughs in next-generation sequencing technologies and associated low-costs in sequencing, ALE is becoming more popular as a tool for biotechnology.
A critical requirement of this method is the aeration of cultures, traditionally done with a magnetic stir bar mixing a flask inside an incubator to generate a vortex on an orbital shaker platform. However, these traditional methods are impracticable for doing in a high-throughput manner for several reasons:
- Traditional stir plates can only mix one vessel at a time; each additional sample would require its own stir plate.
- As the cultures must be heated, the stirring needs to be inside an incubator. There would be space constraints especially if each sample needed its own stir plate.
- The cultures must be sampled regularly (ideally every 10 – 15 minutes) over a period of 24 – 36 hours. When using a laboratory technician, for practical purposes, cultures are sampled once every couple of hours.
- Orbital shakers are not compatible with Automation Platforms.
By using a robotic liquid handler, the preparation and sampling of the cultures can be automated. However, the aeration of the cultures in a high-throughput manner is still a significant hurdle to overcome, especially when considering the limited space allowed by a liquid handler.
V&P Scientific helped one of our customers develop a high-throughput method for Adaptive Laboratory Evolution. V&P designed and built a combination heater/mixer integrated into the Tecan Evo liquid handler. A graphite heat block, VP 741GZ-64, was used for incubation, allowing for up to 64 tubes (50ml conical bottom) to be used. A Magnetic Tumble Stirrer, VP 710T-SM, was used to stir all 64 samples with a single stirrer unit. Each tube utilized a 24 mm diameter stir disc, and when powered by the tumble stirrer, generated a vigorous vortex to the bottom of the tube, thus assuring the maximum transfer of oxygen into the growth medium.
Our customer has written an earlier white paper to report their findings for Adaptive Laboratory Evolution when using V&P’s magnetic tumble stirrer.
"Our laboratory recently changed from a tube based to tray based method for flow cytometry crossmatch (FCXM). A ‘flick’ wash method is commonly used in a tray FCXM; this technique may create potentially biohazardous aerosols in the laboratory. In our experience, technologist to technologist variation is a concern with the flick wash method. We investigated the use of the VP 177A-1 Aspiration Manifold for FCXM tray wash steps and performed a comparison between the flick wash method and this vacuum manifold wash method."
Setting up to use Mag Beads in an application?
Do You Fit Microplate to Magnetic Bead Separation Block or Block to Microplate?
Usually customers come to us with a microplate in hand and ask us which of our MagSep Blocks will work. Most of the time it’s an easy match, but sometimes they have selected that one microplate that is hard to fit any of our blocks or those from other vendors! Let’s look at it from the other way, select the MagSep Block and then the best plate. A cart and horse analogy would fit nicely here, but I’ll restrain myself!
96 Deep Well Microplates and Magnetic Bead Separation Block VP 771MDWM-1
As an example, you know you want a 96 deep well plate (96 DWP) and the strongest of our MagSep Blocks for 96 well microplates, such as the VP771MDWM-1 (the 13-bar magnet block). The shape of the well at the bottom, where the magnetic beads will be collected, and how it is positioned to the bar magnet are the most important considerations. All of these considerations can be affected by the way the Microplate is shaped underneath.
If you like the 13-bar magnet block VP 771MDWM-1 for its separation speed and pellet collection to both sides of the well, a good well shape is the regular v-bottom. This “regular” V-shape helps to position the beads away from the well bottom, similarly to how the steep V-shape bottom does (see below). Plates with the regular v-bottom are usually a 2.2 ml DWP with the square well. Examples of these 96 DWPs are from Costar (3961), Axygen (P-2ML-SQ-C) and Eppendorf (DW96/2000ul). There are probably others as well.
A plate like the Abgene 96 DWP (AB-0932), a 2.2 ml DWP with the square well that has a “steep” V-shape to the bottom part of the well, are the best for the 13-bar magnet MagSep Block to collect the beads in 2 pellets on opposite sides of each well positioned a good distance from the well bottom. Unfortunately, this plate has plastic protrusions underneath -- these are mold “gates” from the injection molding manufacturing process -- that get in the way. So an alternate MagSep Block is the VP 771MWM-1 (the 7-bar magnet block).
It’s not as fast as the VP 771MDWM-1 but still very capable at separating mag beads from a solution to the right or left, alternately, in the wells.
Use the VP 771MCWM-1 (6-bar magnet block) for plates that have a central mold gate. As of yet, I have not found a plate that has the well shape of this Abgene 96 DWP but without the mold gates underneath. Please let us know if you know of one!
And finally, a 96 DWP with a round bottom well also works, but occasionally the collected beads may not be far enough from the bottom center of the well where your pipette tip will be for aspirating the liquid. This can happen if the concentration of beads in the solution is high or if the beads don’t stay in a tight pellet on the side of the well (most likely due to the beads not being very magnetic). These are both situations we have encountered when testing bead separation for customers in order to create as new MagSep Block. So if you find this to be the case for your assay, consider a DWP with a the V-shaped well bottom.
For a longer list of 96 DWPs that have been checked for a good fit on V&P’s 13-bar magnet block (VP 771MDWM-1) and 6-bar magnet block (VP 771MCWM-1) please see this product note:
The plates are listed first according to well volume and then well shape. Where a DWP doesn’t fit either MagSep Block, an alternate one is listed, such as the 7-bar magnet block (VP771MWM-If you don’t see your 96 DWP on the list, send it to us and we will check it for you!
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 710D3 Multi Stirrus™. This design is well suited for mixing different solutions or dissolution of powders.
When used in conjunction with the VP 710D3 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.
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 5 ul droplets dispensed from a liquid handling robot equipped with a 384 channel pipet head; the droplets were dispensed onto a microplate lid.
The liquid handling robot aspirated 1.96 ml media from the VP 534-ALL reservoir which had been filled with only 4.5 ml of starting material. The droplets were consistent in size and the overall weight of the plate before and after dispensing indicated that ~5.1 ul was delivered to each spot:
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 exacting 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 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
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.
A Guide for Selecting the Right Pin for Your Application
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.
5. What is the Z clearance on the robot deck from the highest impediment, top of the source plate, recipient plate, wash reservoirs, etc., to the top of the robot mounting plate? If it is greater than 77 mm you can use any of pins. If it is less than 77 mm, you can only use the shorter FPC series pins 12 mm exposed pin length, the FPN series pins 17 mm exposed pin length and the E-clip series pins 23 mm exposed pin length pins. If the Z clearance is less than 60 mm, we recommend you contact us. There are a few tricks we can do to shave off 5 to 10 mm in height.
6. Is the source plate a deep well plate? If so you may need to use a 30 mm long exposed length pin ("T" pin) to reach down to the lower levels of the well.
7. Should you choose the solid pin versus the slot pin? Although there is a slight advantage for slot pin, CV's between the two pins are very good. Both are easy to clean between specimens. Biggest factor is cost. If you don't need to deal with varying liquid heights and you can obtain the volume necessary with a solid pin, choose the economical solid pin.
8. If you are still uncertain about which pin to select for your application, you can perform a simple "Proof of Principle" test with several different pins using our inexpensive VP 450FP3 Replicator Strip coupled via a VP 452MP to one of our robot mounting plates. An even simpler solution is to use a work station with a 1, 4 or 8 probe dispense head with the V&P Mono Pin Tool to test the various pins.9. If you still have questions you can always contact on one of our friendly technical specialists to help.
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.
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).
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.
V&P Scientific has recently added a new line of stir elements which are capable of re-suspending cells or particles and keeping cells or particles in suspension without causing cell damage.
The VP 772FN-25-55TF-150 (Tuning Fork) and the VP 772FN-20-21CV-150 (Torpedo-Cross) were both designed to be autoclavable. They fit inside 50ml centrifuge tubes to mix the constituents. The outer shape of both stir elements matches the inside topography of the tube. Both designs allow you to take samples while the tube is being stirred. The Torpedo-Cross design has a lower profile and therefore requires less dead-volume than the tuning fork design. Both stir elements use two VP 782N-12-150 (150°C) Neodymium Iron Boron magnetic discs.
These stir elements have optimal performance with our VP 710D3 Multi Stirrus™. Both designs are well suited for mixing different solutions, resuspension of cells, or dissolution of powders. When these stir elements are used in conjunction with the VP 710D3 Multi Stirrus™ more than 24 50ml conical tubes can be mixed simultaneously. The stirring action does not shear or damage fragile cells even after continuous mixing.
In a test experiment using human promyelocytic leukemia cells (HL60), 35ml samples were removed from a tissue culture flask and placed into three different 50ml conical tubes.
The 1st tube was allowed to sit at room temperature with no stirring. The 2nd tube was stirred at 350 RPM with the Tuning Fork design
(VP 772FN-25-55TF-150). The 3rd tube was stirred at 350 RPM with the new Torpedo-Cross design (VP 772FN-20-21CV-150).
The cells in each tube were counted within 5 minutes of being removed from the tissue culture flask. They all had comparable cell counts with percent viability greater than 98%, as measured by Trypan blue exclusion. The three tubes were then allowed to incubate at room temperature for approximately one hour, with 2 tubes being continuously stirred at 350 RPM during the incubation.
The cell counts taken from the tubes being stirred remained consistent with the counts that were taken within the first 5 minutes of removal from the tissue culture flask. The counts from the tube with no stir element showed a drop of 40% due to the settling of the cells.
The three tubes were then centrifuged for 5 minutes at 500 RPM to pellet the cells. The 2 tubes with stir elements were returned to the stir device and stirred for 5 minutes in an attempt to resuspend the pelleted cells. The counts from the tube with no stir device dropped by over 99%, consistent with the pelleting of the cells. The tubes being stirred returned to the counts comparable to those taken within the first 5 minutes of removal from the tissue culture flask. There was no evidence of cell damage seen in any tube as measured by Trypan blue dye exclusion; the percent viability remained greater than 98% in all three tubes.
In a second test experiment, both stir elements were used to mix heavy glitter particulates. The Torpedo-Cross style element was superior in keeping heavy particulates in suspension. When very light particulates (fine glitter) were tested, the Tuning Fork design was equally effective. The Torpedo-Cross stir element, being a two piece design, is more expensive than the single piece Tuning Fork stir element, but offers a greater performance when stirring heavy particulates.
In summation, both the Torpedo-Cross and the Tuning Fork style stir elements provide a safe and effective way of keeping cells or particulates in a uniform suspension throughout a 50ml conical tube, all the while allowing you to take samples while the tube is being stirred. The Torpedo-Cross is superior when using heavy particulates or clumping cells.
This video shows a comparison of the two designs using heavy glitter particulates to simulate heavy insoluble particulates in 40 ml of water.
The new Single-Cell RNA-Seq (RNA Sequencing) protocol called “Drop-Seq” uses V&P Scientific’s Magnetic Tumble Stirrer (VP 710D3), Support Stand (VP 710D2-4) and Magnetic Stir Discs for stirring in a syringe on a syringe pump.
A very interesting paper was recently published in Cell titled, “Highly Parallel Genome-Wide Expression Profiling of Individual Cells Using Nanoliter Droplets,” by Evan Macosko et al in the Steve McCarroll lab at Harvard.
As a former bench scientist who has done her share of gene expression analyses the really old fashioned way, from large numbers of cells of singular origin, like a culture flask of yeast, I can attest that this Drop-seq method is a powerful system these researchers have developed to study complex mixtures of cells at the single cell level.
Read their article to see how the Drop-seq method can be used in your own research (see also their Drop-seq website).
I will explain the use of V&P Magnetic Tumble Stirrer in the Drop-seq protocol to ensure proper setup & use for fellow researchers.
Here is a link to the protocol that the Harvard McCarroll Lab has put together.
I would follow what the researchers have outlined in their protocol for the mixing of the beads. However, I would like to add some details to their setup description. They have a nice diagram of the relative positions of the syringe pumps and the microscope, but it does not include the position of the VP 710D2 relative to the syringe pump it is stirring. They do describe putting the syringe pump in a vertical position such as this:
But what they don’t mention is the VP 710D2-4 Support Stand, which was designed for use when mixing in syringes on syringe pumps. The Stand positions the VP 710D2 above the syringe (or next to it) to allow for more effective stirring by keeping the Magnetic Stirrer away from the pump.
The magnetic stir bars mentioned for use within the syringe are actually stir discs! The thin, Parylene-coated Neodymium discs work well at a distance and do not interfere with the dispensing of fluid from the syringe. They are autoclavable, and priced so that they can be disposable. See our webpage for more information on these and other magnetic stir discs from V&P Scientific.This combination of V&P's innovative magnetic Tumble Stirrer and unique magnetic Stir Discs assisted Evan Macosko and the McCarroll lab at Harvard develop a method that, to paraphrase the title of their paper, profiles the genome-wide expression of individual cells on a large scale. We would love to help you do something similar with your research!
We make stir devices that create a powerful vortex and the centrifugal forces are strong enough to keep magnetic beads suspended.
Our part: VP 776-8-50DM is a demagnetized, round, PTFE encapsulated stir bar (Alnico) 50 mm (+/- 5 mm) long & 8 mm (+/- 0.8 mm) in diameter. When used in combination with the VP 710D2 Multi Stirrus™, your magnetic beads will remain in uniform suspension. They do not attach themselves to the stir bar, and the centrifugal forces keep them from settling to the bottom of the bottle.The same approach can be used for mixing magnetic beads in automation-friendly reservoirs with the use of our stainless steel bubble paddles. The video below shows magnetic beads being resuspended: