Section10:Flipr Instrument Set-Up

From Assay Guidance Wiki

Contents 1 Pre-Assay Setup for FLIPR™-2 and -3 2 Sequences Setup 3 Post Assay Setup 4 Graph Setup 5 One-, Two- and Three-Addition Assay Examples

Pre-Assay Setup for FLIPR™-2 and -3

In this screen, which is the same for 96- or 384-well assay set-up, the user defines the labware used in the experiment from a drop-down list. The other options on this screen are the filter selection, camera configuration, and the output file setup.

1. Assign Plate: This is where the user configures the deck layout. If the plate you are using is not included, there is a default 96-well and default 384-well that can be used until the correct plate is defined.
2. Camera configuration: The exposure length is typically set to 0.4 secs. The gain is only applicable to The FLIPR™-3 with the Andor camera.
   
   Note: To adjust the baseline signal of the plate, first adjust the laser intensity from the keypad before adjusting the exposure time. This should be done for each plate to set the same baseline over a run.
3. Filter selection: The FLIPR™ has a two-position filter slide. Typically, filter #1 is a 488-nm filter used for calcium assays, and filter #2 is either blank or a 535-nm filter for membrane potential assays.
4. Create document name: This is where the filename is created. A “1” in the field means this will be included in the file name and a “0” means it will not. A few issues deserve a warning here: If you use the date only, it is very possible that the data generated will be overwritten if another run is made on the same date. Therefore, it is a good practice to include a user-defined string in your file name. ALWAYS include the experiment number in the output. This is the flag that assigns the _n1,_n2, etc to the plates in the run. Failure to include this will result in every plate being labeled _n1, thereby overwriting all previously generated data. The best practice here is to use a lab notebook number and page as the filename. An example would be: D00567_143, where D00567 is the notebook number and 143 is the page.

Sequences Setup

The sequence setup is where the entire experiment is defined. This includes defining the number of reads to be taken as well as all liquid handling steps, wash sequences, automated tip unload, etc. These settings should be done with the assistance of an automation engineer or an experienced FLIPR™ user.

By double clicking on the circle beside each step, the user can activate/deactivate that part of the sequence. A green circle indicates the step is active while grey indicates inactive. By single clicking on the sequence step, the step’s setup box appears on the right side of the window with all parameters that can be accessed by the user.

1. Pre-Soak: This is typically not used.
2. Aspirate: The FLIPR™ can aspirate from any of the four deck positions as long as a plate has been defined there in the initial setup page.
3. Put tips in target well: This will move the tips into the target plate before dispensing. Typically not used. NEVER use this if dispensing at a low height where the tips are in contact with the buffer. We have observed that this can cause a response from the compound on the outside of the tips.
4. Baseline imaging: The pipettor head will not move to the cell plate until the baseline imaging is complete. A typical setting is 1 to 5 secs.
5. Dispense: The FLIPR can dispense to any of the four deck positions as long as a plate has been defined there in the initial setup page.
6. Wash tips: This will wash tips in the wash station at position 6 if the unit has a wash station installed. A pre-wash can be performed at position 5 by clicking the “rinse after wash” button. This will use the same wash parameters defined, only perform them at position 5.
7. First Interval: This sets the number of images to be acquired and the interval between each image. Typically, the interval is short (1 sec) and the number of images are 30 to 60 to capture the compound addition. This should be set long enough to capture past the peak response.
8. Second Interval: This set the number of images to be acquired and the interval between each image. Typically, the interval time is longer (3-5 secs) and the number of images is sufficient to capture when the response decreases to background. In some cases, the signal will never return to background and it is the judgment of the scientist to set this range.
9. Automated Tip Unload: This will automatically unload the tips to the rack when all pipetting steps are completed. This should only be done in the last sequence.
10. Clear Pipette Head: This return the pipettor head to the home position.

Post Assay Setup

In the post-assay setup section, the user selects where data will be saved, what type of data to save, and considers the option to automatically export and print data at the end of each plate. When setting the save location, you must type in the exact path to the save directory. The software will generate an error if the location is invalid or if it is a network location that is not available. In most instances, only FWD files should be saved. This saves storage space, as the FID files are larger image files. In some instances, such as when a heated stage is used, the open door may need to be turned off to maintain better temperature control in the FLIPR™.

Graph Setup

Typically, Spacial Uniformity Correction is used without subtracting the background. Spacial Uniformity Correction is basically a software normalization that sets all wells to the average RFU of the plate when starting the experiment.

In most cases, subtract bias is not used. This will background subtract the data set which can mask the assay window. An example would be to start with a baseline of 5000 RFU and the max signal response being 6000 RFU. In most situations, this is not a screenable window, but if the 5000 RFU background is subtracted, the window “looks” good (0 to 1000).

One-, Two- and Three-Addition Assay Examples

All three of these formats will require the same initial setup described above.

One-addition assays will need one or two sequences dependent upon the use of an in-tip dilution. The example below shows a 384-well aspiration from position 3 with a dispense into the cell plate at position 1 (Read Position), followed by a wash.

A one-addition assay with an in-tip dilution is shown below. The first step aspirates 17µl from plate 1 and then 8µl from plate 3.

Note: When performing an in-tip dilution, the volume in the second step is the final total volume aspirated (17µl + 8µl). This is a result of the way the FLIPR™ software keeps track of the pipettor head.

A two-addition or three-addition assay can be run by simply adding sequences. It is recommended that if the assay is targeting potentiators, the in-tip dilution and pre-incubation time be used to maximize the sensitivity of the assay. Below is the complete liquid handling setup for a three-addition assay. Volumes and read times will vary.

Note that in sequence 3 and 5, the order of aspiration is reversed. This is due to the fact that unknown test compounds have been added to the cell plate and to aspirate from there first would be a source of contamination to the EC10 reservoir. This is not the case for the 4th and 6th sequence as the tips have been washed.