Basic Protocol for the SciFlow™1000 System Create Fluorescein Standard Curve Calibration for Flow Tracking Drug Exposure & Monolayer Culture

System Description

The SciFlow™ 1000 Fluidic Culture System is a benchtop tool for in vitro use to mimic cell, organ, and living systems. The SciFlow™ System operates like a shallow river bed with a series of compartments for cell culture. The design allows for isolated and stagnant culture during cell seeding, and then delivers real-time fluid flow and compartment-to-compartment signaling over time. The entire system is contained within a 96-well formatted culture plate that includes 8 repeatable channels. Each channel has the capacity to connect 1-to-10 cell culture wells in a linear array. As a benchtop tool, the SciFlow™ System is configured for cell and tissue assessments allowing easy access to all culture wells and media streams. Additionally, the SciFlow™ System is compatible with microplate readers, high content imaging platforms, and microscopes.

Benefit: No external pumps, tubes, nor controllers are required.

Protocol Focus: There are many applications of the SciFlow™ 1000 System. This protocol focuses on creating a fluorescein standard curve and subsequently estimating the dilution of test compounds across a SciFlow™ channel. Fluorescein is used as a tracer dye to extrapolate concentrations of compounds (concentration vs. time) across linked cell compartments as fluid moves across a channel.

Once the standard curve is generated, a line equation is calculated. This equation will allow back calculation of an absolute fluorescein concentration which is then correlated to the starting concentration of the drug or toxicant.

In order for this approach to be effective, users must first optimize gain settings for the highest concentration of fluorescein (recommended starting concentration 1–10 µM). The starting concentration will vary depending on instrument sensitivity and settings. Then, users must manually set the gain levels to be the same for each subsequent experiment.

Reagents and Supplies:

  1. 100 µM Fluorescein: Make a stock solution by dissolving fluorescein sodium salt (Product Number F6377) in the base medium without supplements.
    1. Example: If the base medium is DMEM, dissolve fluorescein in DMEM to 100 µM. Protect from light and maintain sterility. This stock solution can be stored at 4 °C wrapped tightly in foil.
  2. One SciFlow™ 1000 System.
  3. Culture medium - This is the medium used to culture the cells. If different media are used for different cells, a standard curve calibration will need to be performed for each medium.
  4. CAUTION!! Check the height limitations on the plate reader to ensure the SciFlow™ plate will fit. Call the manufacturer with the height values. See Key Dimensions below for vertical height confirmation.

Key Dimensions for SciFlow™ Plates

Key Dimensions for SciFlow™ Plates



  1. Create a dilution curve from 1 µM to .001 µM Fluorescein in 2 mL dilutant. When creating dilutions, dilute into complete culture medium (Base medium plus any serum or additives). Always protect from light as much as possible. Fluorescein rapidly quenches when left exposed to light.
  2. Remove a sterile SciFlow™ System from packaging.
  3. Load one concentration of dye along an entire row, 100 µL per well with 500 µL in the source well. For example:
    Row A: 1 µM
    Row B: 0.3 µM
    Row C: 0.1 µM
    Row D: 0.03 µM
    Row E: 0.01 µM
    Row F: 0.003 µM
    Row G: 0.001 µM
    Row H: Medium only, no dye
  4. Set fluorescent plate reader instrument to optimize gain based on 1 µM Fluorescein. Read plate at excitation wavelength = 485 nm, emission wavelength = 520 nm
    1. Coefficient of variance across each row should be calculated from data. If the plate reader allows adjustment of z-height focal length, make z-height adjustments until CV is at lowest point. See Key Dimensions for SciFlow™ Plates for specifications to aid in creating appropriate z-height settings.
    2. IMPORTANT: Note the gain level that the instrument chooses for 1 µM Fluorescein. This will be the gain level that must be used for all subsequent experiments.
    3. For some instruments, the gain is preset. Call the manufacturer to clarify how to determine and set the gain appropriately.
  5. Plot data by averaging fluorescent units across rows against concentration of fluorescein.
  6. Create a line equation that will be used to predict fluorescein concentrations based on fluorescent units collected at the gain level developed in step 5.
  7. Record this line equation with the gain setting for future use.
  8. With new stock reagents, changes in culture medium, or from time to time, recalibrate the gain level by using a new plate and re-optimizing gain based on 1 µM Fluorescein. Always store equation and gain levels together for accuracy.


Fluorescein Standard Curve Example

Below is an example of creating a Fluorescein standard curve. In this example, a Tecan Infinite M1000 Pro was used to generate RFU. 100 µL of each dilution was added to all the wells of a single row. The average and standard deviation was used to create a line equation for subsequent experiments.

Mode Fluorescence Top Reading
Excitation Wavelength
Emission Wavelength 525  
Excitation Bandwidth 5  
Emission Bandwidth 5  
Gain 88  
Number of Flashes 10  
Flash Frequency 400 Hz
Integration Time 20 µs
Lag Time 0 µs
Settle Time 0 ms


Raw Data

µM Fluor-escein 2 3 4 5 6 7 8 9 10 11
1 61722 62521 63000 63000 62569 62916 62620 62820 62364 63000
0.3 18967 19849 20243 21107 20361 21105 20220 20112 20182 21087
0.1 6917 7468 7669 7535 7633 7673 7720 7696 8677 6787
0.03 2683 2952 2886 3044 3121 3008 2953 3021 2948 3049
0.01 1437 1580 1507 1417 1505 1548 1621 1493 1574 1628
0.003 1055 1063 1040 1056 1042 1017 1026 1057 980 1051
0 806 799 803 782 811 818 793 744 762 726


Summary Stats

µM Fluorescein Average Stdv %CV
1 62653.2 399.2 0.6
0.3 20323.3 663.0 3.3
0.1 7577.5 509.8 6.7
0.03 2966.5 119.7 4.0
0.01 1531 71.9 4.7
0.003 1038.7 25.2 2.4
0 784.4 30.7 3.9


Graph and Line Equation


Sample Plate Map

Sample plate map

Example of Cell Seeding Parameters
SciFlow™ culture well areas are ½ the size of traditional 96-well culture surface areas (0.167 cm2 or 16.7 mm2).

Cell Seeding Examples, 2D monolayers Number of Cells per SciFlow™ Plate Number of Cells per Well Number of Culture Wells Seed Time Initial Confluence Adjustment
Primary human hepatocytes with collagen coating 2.0 E6 27,500 72 (3 – 11) Overnight Confluent By viewing
Primary rat hepatocytes with collagen coating 1.0 E6 14,000 72 (3 – 11) Overnight Confluent By viewing
Primary mouse hepatocytes with collagen coating 6.5 E5 9,000 72 (3 – 11) Overnight Confluent By viewing
Primary duck hepatocytes with collagen coating 6.5 E5 9,000 72 (3 – 11) Overnight Confluent By viewing
Primary canine hepatocytes with collagen coating 1.0 E6 14,000 72 (3 – 11) Overnight Confluent By viewing
HepG2 2.2 E6 30,000 72 (3 – 11) Overnight 80% By viewing
HepaRG 2.9 E6 40,000 72 (3 – 11) Overnight 80% By viewing
HepaRG (no spin) 2.9 E6 40,000 72 (3 – 11) Overnight 80% By viewing
Cell Line MCF7 7.2 E5 10,000 72 (3 – 11) Overnight 20% By viewing


Removing Medium (if required): The SciFlow™ System can be emptied by inversion and flicking into an appropriate waste container. Additionally, the entire row can be emptied via vacuum aspiration through the sink well.

Moving the SciFlow™ 1000 System: The SciFlow™ System is a fluidic system and tipping the plate can disrupt or modify both flow and any established gradients. Reasonable care should be taken when moving the plate to minimize unintended flow caused by tipping the plate.

Evaporation: Alhough the SciFlow™ 1000 System does have a lid, evaporation can occur. For experiments over 7 days, a 10–20% greater volume can be added to the source well than is removed from the sink.