96-inserts with differentiated BCRP-expressing MDCKII cells
USER’S MANUAL (Corning Plate)PreadyPort™ BCRP 96-well – Corning plate - User’s Manual

Limited Single-use LicenseThis is a binding legal agreement (this “License”) between you (“Customer” or “You”), andSOLVO and READYCELL regarding the enclosed PreadyPort™ System (this “Product”). Carefully
read this License prior to opening the sealed package. BY OPENING THE SEALED PACKAGE, YOU
AGREE TO BE BOUND BY THE TERMS OF THIS LICENSE. If You do not agree to the terms of thisLicense, and You have not opened the sealed package, promptly notify both READYCELL and
SOLVO in writing and return the unopened Product and all accompanying items (including all
written material and containers) to READYCELL within five (5) business days.
This License sets forth Customer’s rights to use this Product. The meaning of “Product”
hereunder shall include any component parts thereof and any documentation made available
for use with this Product. Upon receipt of the applicable fee, SOLVO and READYCELL grant toCustomer the personal, non-exclusive, and non-transferable right to use this Product inEurope/USA. Customer is solely responsible for the use of this Product by any person.
Customer acknowledges and agrees that the manufacture, use, sale or import of this Productmay be subject to one or more issued or pending patent applications and corresponding
foreign equivalents owned or controlled by SOLVO and READYCELL. Customer acknowledges
and agrees that the documentation and components included in the Product are the
proprietary and copyrighted material of SOLVO and READYCELL. The purchase and use of this
Product shall be governed under SOLVO’s and READYCELL’s standard Terms and Conditions,and any other written agreement between the parties, and such terms and agreements areincorporated herein by reference. Your purchase of this Product, subject to this License,
conveys to Customer the non-transferable right to use the purchased amount of the Product inthis shipment and components of the Product in this shipment in research conducted by
Customer, such right to expire upon the completion of experiments from this shipment.
Customer shall not detach living cells from the product and shall not amplify genetic material(DNA, RNA) for purposes other than quantitative or qualitative analysis of proteins or
nucleotide sequences.
Customer shall not sell or otherwise transfer (a) this Product (b) any components of this
Product; or (c) any materials made using this Product or its components, and Customer shallnot otherwise use this Product or its components for Commercial Purposes as defined herein:(1) use of this Product in manufacturing; (2) use of this Product for therapeutic, diagnostic orprophylactic purposes; or (3) resale of this Product, whether or not such Product is resold foruse in research. For clarification, Customer may use and transfer information or materials
made through the use of this Product to support Customer’s research in any manner Customer
deems necessary or appropriate and Customer may provide services to third parties.
Subject to the terms of this License, SOLVO and READYCELL will not assert a claim against
Customer alleging infringement based upon the manufacture, use, or sale of a therapeutic,
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Product the manufacture of such Product.
If you have any questions or you need information on obtaining a license to use this Product orany components for purposes beyond the scope of this License please contact SOLVO or
READYCELL on sales@solvo.com or helptech@readycell.com, respectively.

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PreadyPort™ BCRP 96-well – Corning plate - User’s Manual

TABLE OF CONTENTS
Intended Use .......................................................................................................................................... 3
Principle .................................................................................................................................................. 3
Timelines for Delivery and Experimental Procedures .......................................................... 3
Equipment (Not Included) ............................................................................................................... 4
Consumables .......................................................................................................................................... 4
Material Not Included ........................................................................................................................ 5
Unpacking and Maintenance ........................................................................................................... 6
Unpacking .......................................................................................................................................... 6
Replacement of Shipping Medium............................................................................................ 6
Replacement of Cell Culture Medium ...................................................................................... 7
Induction of Protein Expression................................................................................................ 7
Quality Control of the Barrier System.......................................................................................... 8
1. Pre-assay Quality Control - Measuring TEER .................................................................. 8
2. Post-assay Quality Control - Lucifer Yellow paracellular permeability assay. ... 9
General Protocol for Transport Assays .....................................................................................10
Recommended Controls .............................................................................................................10
Apical-to-Basal studies ...............................................................................................................10
Basal-to-Apical studies ...............................................................................................................13
Evaluation of the Test Compound Transport .........................................................................15
Normal Range of Controls (According to FDA, p. 64 Fig. A1) ...........................................15

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PreadyPort™ BCRP 96-well – Corning plate - User’s Manual

Intended Use
The contents of this product are designed to conduct monolayer transport experiments.
Monolayer assays may be used for the evaluation of interactions with drug transporters andalso to model pharmacological barriers.
This kit is optimized for interaction studies with the human breast cancer resistance protein(BCRP, MXR, ABCG2).
Handling and experimental procedures are provided below. The manual has been written forusers with experience in cell culturing and monolayer transport experiments. For moredetailed advice and training opportunities please contact:
support@solvo.com, or
helptech@readycell.com
The kit is intended for in vitro purposes only. Not for human or veterinary use.

Principle
In the monolayer transport experimental setup, two solute compartments are separated by atight cell monolayer. The distribution of compounds between the two compartments isdetermined by qualities of the cell monolayer, most notably the presence of transporter
proteins in the cell membrane.
The differentiation of the cells defines an apical and a basolateral solute compartment as wellas the localization of transporter proteins. Apically localized efflux transporters will introduce abasolateral-to-apical bias in the distribution of substrate compounds.
In a standard assay design, the reaction is initiated by filling either compartment with thesolution containing the test compound. The distribution is assessed over time by withdrawing
and analyzing samples from both compartments. After normalization the speed oftranslocation is obtained in both directions. The ratio of the two values is a measure of active
processes involved in the distribution of the compound.

Timelines for Delivery and Experimental Procedures•

Day 1: Starting production (Seeding of cells)•

Day 4: Pre-shipping quality control (TEER and Lucifer Yellow)•

Day 5: Package Dispatch•

Days 6-7: Package Delivery•

Day 8: Replacement of Shipping Medium•

Day 11: Quality Control Experiments, Medium Replacement•

Days 12-15: Transport Experiments

Packages are dispatched on Tuesday and are delivered within two days in EU countries. For
other locations and customized schedule please contact us at
support@solvo.com, or
helptech@readycell.com+ 34 93 403 70 77
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PreadyPort™ BCRP 96-well – Corning plate - User’s Manual

MondayTuesday

WednesdayThursdayFridayDay 1

Week 0Saturday

Sunday

Day 2

Day 3

Day 9

Day 10Starting production
Day 5

Day 4

1st Week

2nd Week

Pre-shipment
Quality controls
Day 11
Measuring TEER andreplacement of cell
culture media

Day 6

Shipment of plates
Day 12

Day 7

Liquefaction oftransport medium

DeliveryDay 13

Day 8

Day 14Day 15

Days to perform your assays (Apply and Assay)

Figure 1. Timeline of manufacturing and operation for PreadyPort™ BCRP 96-well kits

The recommended timing for transport experiments is Day 12 (Tuesday). If you choose to
conduct the experiments later, medium must be replaced as follows:
DAY OF EXPERIMENTMEDIUM REPLACEMENT

Tuesday (Day 12)

Monday (Day 11)

Wednesday (Day 13)

Monday (Day 11)

Thursday (Day 14)

Monday (Day 11) and Wednesday (Day 13)

Friday (Day 15)
Monday (Day 11) and Wednesday (Day 13)
Table 1. Recommended days for medium replacement

Equipment (Not Included)•

Cell culture laminar flow hood•

CO2 incubator•

Aspiration system•

Multichannel pipettes•

96-well format vacuum manifold (Drummond, cat. No 3-000-093 recommended)•

Trans-Epithelial Electrical Resistance (TEER) meter (Ref Millicell© ERS-2 or WPI
EVOM2)•

Recommended 96-well electrode (Ref WPI STX100C96)•

Quantitative analytics equipment

Consumables•

Reservoir plate (Provided by ReadyCell)•

Pipette tips (Not provided)•

Sterile culture medium containers Ex. Costar 50 ml #4870 (Not provided)

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PreadyPort™ BCRP 96-well – Corning plate - User’s Manual

Material Not Included•

MDCKII Cell Culture Medium. Dulbecco’s Modified Eagle Medium with the following
supplements (final concentration):o

5 mM Glucoseo

25 mM HEPESo

10% V/V Fetal Calf Serumo

2 mM L-glutamineo

100 U/ml; 0.1 mg/ml Pen/Strepo

1x MEM non-essential amino acid solution•

Cell Culture Medium for Induction of BCRP expressiono MDCKII Cell Culture Medium supplemented with 1 mM sodium butyrate(Sigma-Aldrich, 303410)•

Assay Buffer

For the preparation, dissolve the following compounds in 800 ml purified water (drypowder weight):o

4 .8 mM KCl (0.36 g)o

0.96 mM KH2PO4 anhydrous (0.17 g)o

1.2 mM MgSO4·7H2O (0.3 g)o

12.5 mM HEPES (3 g)o1.53 mM CaCl2 (0.17 g)o

142 mM NaCl (8.3 g)o

5 mM glucose (1 g)

Adjust pH to 7.4 with 1 M NaOH.
Bring total volume to 1000 ml and filter the solution using a 0.22 μm sterile filter.
Buffer can be stored at 2-8°C for a maximum of 1 month after sterilizing.
•

Recommended reporter substrate: 1 µM Prazosin (Sigma-Aldrich, P-7791) in DMSO•

Recommended Inhibitor: 1 µM Ko143 (SOLVO-Biotechnology, SB Ko143) in DMSO

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PreadyPort™ BCRP 96-well – Corning plate - User’s Manual

Unpacking and MaintenanceUnpackingUpon reception retrieve the zip-top bags containing the plates. Open the zip, and leave the bag
at a dark location at room temperature until Day 8 (refer to Timelines).

Replacement of Shipping Medium
Caution: Never handle more than one plate at a time while changing the shipping medium. Resolidification of the shipping medium may provoke mechanical damage to the cell monolayer.
These steps have to be carried out on Day 8 (refer to Timelines and see Figure 2). (Perform all
manipulation under aseptic conditions).
1. Retrieve the plates from the bags and remove the parafilm wrap.
2. Incubate the plates in a 5% CO2 humidified atmosphere at 37°C for 4 hours.
3. Remove one PreadyPort™ plate from the incubator and place inside the laminar flow
hood, along with one reservoir plate.
4. Using sterile procedures (inside the laminar flow hood):- Fill a sterile reagent reservoir with 50 ml of pre-warmed (37°C) MDCKII culture
medium.
- Open the PreadyPort plate and the reservoir plate, and leave the lids upwards, next
to the plates.
- Carefully lift up the 96-integrated apical compartments of the PreadyPort™ plate
and transfer onto the reservoir plate.
- Remove all liquefied shipping medium from the basal compartments of thePreadyPort™ plate via aspiration with the 96-well manifold.
- Using a multichannel pipette, dispense 235 µl of MDCKII cell culture medium fromthe sterile reservoir, and fill, column by column, the basal compartments of thePreadyPort™ plate.
- Using the aspiration manifold connected to a vacuum (adjust aspiration flux tomedium-low), aspirate the liquefied shipping medium from the apical integrated
inserts of the PreadyPort™ plate, taking care not to disrupt the monolayer. Check
that the shipping medium has been removed from all wells. Approximately 10 µl of
medium will be left in each well.
- Using a multichannel pipette, dispense 75 µl of MDCKII cell culture medium fromthe sterile reservoir, and fill, column by column, each of the 96 apicalcompartments of the PreadyPort™ plate. Always add the medium against the wallof the well, not onto the center directly on the cell monolayer.
- Carefully return the apical inserts onto the basal compartment of the PreadyPort™
plate. Replace lid and place inside cell culture incubator, set at 37°C, with 5% CO2.

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PreadyPort™ BCRP 96-well – Corning plate - User’s Manual
5. Once shipping medium has been changed to fresh MDCKII cell medium, plates should
be kept inside the incubator until next Monday (Day 11)

Preadyport ™ plate

Reservoir plate

Preadyport ™ plate

Reservoir plate
Remove liquefied
shipping medium

1

5
Add fresh MDCKII
medium2
Remove liquefied
shipping medium

63
Add fresh MDCKII
medium4

7

Shipping
mediumMDCKII
mediumFigure 2. Scheme of the replacement of the shipping medium

IMPORTANT NOTE: Keep the reservoir plate provided by ReadyCell™. It will be necessaryduring the permeability assay.

Replacement of Cell Culture Medium
Cell culture changing have to be carried out once (at day 11) or twice (at day 11 and day 13)
depending on the day of the assay, following the same procedure described in the previous
section (refer to Timelines and see Figure 2). Always work under sterile conditions. If Day 12 is
the set date for transport experiments, these steps have to be carried out with cell culture
medium for induction of protein expression (cell culture medium supplemented with sodium
butyrate), as described in the “Material Not Included” section.

Induction of Protein Expression
The final cell culture medium replacement, which takes place the day before transport
experiment is conducted, must be done with sodium butyrate supplemented Cell Culture
Medium (1 mM final concentration), as described in the “Material Not Included” section. Allprocedure steps are identical to those described in the section Replacement of Cell Culture
Medium.

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PreadyPort™ BCRP 96-well – Corning plate - User’s Manual

Quality Control of the Barrier System
1. Pre-assay Quality Control - Measuring TEER
This section provides general instructions for TEER measurements. It is important to read the
instructions of the TEER measurement equipment carefully in conjunction with these
instructions:
On Day 11 (Monday) perform TEER measurement on the plate/plates before any further
processing.
To proceed with the TEER measurement, follow the steps below:
1. It is highly recommended to use the WPI STX 100C96 electrodes, in order to avoid
any damage in the cell monolayer.
2. Sterilize the electrodes (probe) by submerging both electrodes in ethanol (70%) for 30
minutes.
3. Equilibrate the electrodes (probe) for 30 minutes in MDCKII medium, pre-warmed atroom temperature.
4. While the electrodes are equilibrating, remove the PreadyPort™ plate from the
incubator and place in a laminar flow hood. Allow the plate to reach room
temperature (approximately 20 minutes), as TEER measurements should be performedat room temperature.
5. Insert the probes into the insert system, so the thinner electrode is placed within the
narrowest slit, which corresponds to the basal part of the inserts. The thicker electrodemust be placed inside the widest slit, corresponding to the apical part of the inserts.
6. Record the TEER value for each well. A TEER value of 100 Ω cm2 (or higher) indicatesthat the barrier system is acceptable for a transport assay (the active membranesurface is 0.14 cm2).

KEY DATA
Membrane area
Minimum TEER value

0.14 cm2
100 Ω·cm2*if WT is included in the kit, TEER value has to be ≥ 70 Ω·cm2

IMPORTANT TIPS:•••

TEER reading should be carried out 48 hours after medium changing in order to verify
the integrity of the monolayer prior to the permeability assay. Never perform the TEER
measurement with the shipping medium
Do not repeat TEER measurements on the same insert
For an appropriate TEER measurement, both electrodes have to be submerged wellwithin the cell culture medium both in the apical and basal compartments

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PreadyPort™ BCRP 96-well – Corning plate - User’s Manual

2. Post-assay Quality Control - Lucifer Yellow paracellular permeability
assay.
Prepare a Lucifer Yellow stock solution of 1 mg/ml in sterile ddH2O. Aliquot (for example 500 µl) and store at -20°C.
From the stock solution, prepare a 100 µM working dilution in assay buffer to be used for the
standard curve and the LY test. These dilutions of LY should be pre-warmed (37°C) before
application (see figure 3 for recommended concentrations).
To proceed with the LY permeability assay follow the steps below:
1. Rinse both the apical and the basal compartment gently with assay buffer (75 µl apical/ 225 µl basal).
2. Add 75 µl of 100 µM Lucifer Yellow into the apical compartment.
3. Add 225 µl of assay buffer to the basal compartment.
4. Incubate the PreadyPort™ plate, protected from light, in a 37°C cell culture incubatorfor 1 hour.
5. Recover 200 µl from the basal compartment and dispense into an empty 96-wellfluorometer plate (Mix well and avoid bubble formation when aspirating).
6. Read the fluorescence intensity directly in a fluorometer, a 485 nm excitation filter anda 527 nm emission filter.
Working dilution
100 µM75 µl
200 µl basal

LY + sterile ddH2O
1 mg/ml

Dilutionson HBSS

Reading

Insert200 µlStock solution
Dilutions for standard curve(Table below)

Fluorometer plate forthe standard curve andsamples

Fluorometer

Figure 3: General procedure and recommended concentrations for the standard curve.

Under standard PreadyPort™ conditions the amount of translocated LY should not exceed 2%of initial amount on the donor side. Results over this threshold signals damage to the barrier
incurred during the transport experiment.
KEY DATALY Paracellular Flux
LY Papp≤ 2%≤ 4.5 x 10-6 cm/s

IMPORTANT TIPS:••

Perform the LY Assay after the test compound permeability assay.
It is highly recommended that some of the inserts are reserved and used exclusively
for testing LY permeability, as a quality control test of the monolayer status.
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PreadyPort™ BCRP 96-well – Corning plate - User’s Manual

General Protocol for Transport Assays
Recommended Controls
The following controls are recommended to be included in all plates of the PreadyPort-BCRP
kit:• Active transport (1 µM prazosin, both directions)• Selective inhibition of prazosin active transport (1 µM Ko134, both directions)
The following controls are recommended as optional measures of passive flux:•

Passive transcellular flux (10 µM atenolol or 10 µM metoprolol, apical to basolateral)•

Paracellular flux (100 µM Lucifer Yellow, apical to basolateral)

Apical-to-Basal studies
Test compounds are applied to the apical side of the cell monolayer (upper compartment of
the insert), and the apical-to-basal (A-B) transport through the cell barrier is evaluated bysample recovery and test compound detection in the basal (lower) compartment over adefined incubation period. A-B permeability of test compounds is determined as thecoefficient of apparent permeability (Papp) in cm/s.
The protocol provided below recommends the following conditions for performing transport
assays:••••Test compound concentration: 10 µM
Replicates: 3
Time points: 2 (0 and 2 hours)
Volumes:• Apical compartment: 75 µl of the test compound at working solution (100 µl
is added to the apical compartment and 25 µl is recovered at time 0hr.)•

Basal compartment: 250 µl of HBSS assay Buffer

Note 1: All the following manipulations must be performed under standard sterile conditions used forcell culture
Note 2: Assay buffer should be pre-warmed to 37°C to avoid temperature stress
Note 3: CAUTION, do not add the Lucifer Yellow reagent together with the test compounds. Lucifer
Yellow may interfere with certain substances, producing false results

1- Prepare stock solutions of the test compounds in assay buffer. In case of poorly watersoluble compounds, DMSO may be used at a final concentration of 1% in assay buffer
without significantly affecting the barrier properties.
2- Prepare working dilutions of test compounds in assay buffer.
3- Fill a sterile reagent reservoir with 50 ml of pre-warmed (37°C) assay buffer
4- Unwrap one receiver plate in the laminar flow hood where the experiments will be
performed.
5- Remove one PreadyPort™ plate from the incubator and place it in the laminar flow hood
beside the reservoir plate. Both plates should be oriented the same way.
6- Open the PreadyPort™ plate and the reservoir plate, and leave the lids upwards next to theplates.
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PreadyPort™ BCRP 96-well – Corning plate - User’s Manual
7- Carefully lift the 96 apical inserts of the PreadyPort™ plate and transfer to the reservoir
plate.
8- Remove all MDCKII medium from the wells of basal compartments of the PreadyPort™
plate via aspiration using the 96-well manifold.
9- Using a multichannel pipette, fill, column by column, each of the 96 wells of basal
compartments of the PreadyPort™ plate with 250 µl of pre-warmed (37°C) assay buffer.
10- Remove all MDCKII medium from the apical inserts of the PreadyPort™ plate by aspiration
using the 96-well manifold. Place the manifold perpendicular to the cell monolayer andclose to the insert wall, and take care not to disrupt the cell layer.
11- Using a multichannel pipette, fill, column by column, each of the 96 apical inserts of thePreadyPort™ plate, with 75 µl of pre-warmed (37°C) assay buffer.
12- Carefully return the 96 apical inserts onto the wells of the basal compartments of thePreadyPort™ plate (original position).
13- Incubate the plate for 1 minute at room temperature inside the laminar flow hood.
14- Repeat rinsing the plate (steps 8 to 12), two more times and keep the plate for 30 min a
37ºC inside a 5% CO2, incubator.
15- Take the plate form the incubator, return it to the laminar flow hood and place it next to
the reservoir plate. Both plates should be oriented in the same way.
16- Carefully lift the 96 apical inserts of the Preadyport™ plate and transfer them to the top ofreservoir plate.
17-Remove the assay buffer from the basal compartment of the Preadyport™ plate by
aspiration with the 96-well manifold.
18- Using a multichannel pipette, fill, column by column, each of the 96 wells of the basal
compartments of the PreadyPort™ plate with 250 µl of pre-warmed (37°C) assay buffer.
19- Remove the assay buffer from the apical inserts of the Preadyport™ plate by aspiration
with the 96-well manifold (Place the manifold perpendicular to the cell monolayer and close to the insert
wall, and take care not to disrupt the cell layer).

20- Using a multichannel pipette, fill, column by column, each of the 96 apical inserts of thePreadyPort™ plate with 100 µl of pre-warmed (37°C) assay buffer.
21- Recover 25 µl from the apical inserts (0 hr. Apical samples) and keep them at -20ºC until
analysis.
22- Carefully transfer the 96 apical inserts onto the wells of the basal compartment of thePreadyPort™ plate (original position).
23- Take the plate from the incubator, return it to the laminar flow hood and place it next to
the reservoir plate. Transfer the apical inserts onto the reservoir plate.
24- Recover 25 μl from the apical inserts (2hr. Apical samples) and keep at -20°C until analysis.
25- Recover 25 μl from the basal compartments (2hr. Basal samples) and keep at -20°C until
analysis.
26- Analyze the samples (0 hr. and 2 hr., above).
27- After quantification of test compound within samples, calculate the permeability
coefficient (Papp) as indicated in section “Evaluation of the compound permeability”.

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PreadyPort™ BCRP 96-well – Corning plate - User’s Manual

A to B assay
Test compoundStocksolution

LegendWorking
dilution

Apical compartment
96 Corning plate

Dilution

Basal compartment
96 Corning plateon assay buffer
Reservoir plate

10 µM
Aspirate

1 min 1 st time
1 min 2 nd timeX3
30 min 3 rd time

Fill up withbuffer
250 µL100 µL0h Apical
sampleAspirate

Fill up withbuffer2h Basal
sample25 µL

Fill up withworking dilution

2h Apical
sample
25 µL

25 µL

Incubate 2 h at
37⁰ C, 5% CO2

Cell culture
mediumAssay buffer

Assay conditions
Assay volumes•Apical: 75 µl
2Membrane area: 0.14 cm•Basal: 250 µl
Replicates: 3

Concentration: 10 µM

Time points: 2 (0 and 2 hours)

Sample volume: 25 µl

Working dilution

Sample for analysis

Quality control parameters
TEER: >100 Ω cm2
Lucifer Yellow•Paracellular flux: 2%•Papp : ≤ 4.5 x 10 -6 cm/s

Figure 4: General procedure for A to B assays.

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PreadyPort™ BCRP 96-well – Corning plate - User’s Manual

Basal-to-Apical studies
Test compounds are applied to the basal side of the cell monolayer (lower compartment of theinsert), and their basal-to-apical (B-A) transport through the cell barrier is evaluated by sample
recovering and test compound detection at the apical side (upper compartment) over adefined incubation time. B-A permeability of test compounds is determined as the coefficientof apparent permeability (Papp) in cm/s.
The protocol provided below recommends the following conditions for performing transport
assays.
••••Test compound concentration: 10 µM
Replicates: 3
Time points: 2 (0 and 2 hours)
Volumes:• Apical compartment: 75 µl of HBSS assay Buffer• Basal compartment: 250 µl of the test compound at working solution (275 µl
is added to the apical compartment and 25 µl is recovered at time 0hr.)

All other steps are identical as in the AB protocol, above.

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PreadyPort™ BCRP 96-well – Corning plate - User’s Manual

B to A assay
Test compoundStocksolution

LegendWorking
dilution

Apical compartment
96 Corning plate

Dilution
Basal compartment
96 Corning plateon assay buffer
Reservoir plate

10 µM
Aspirate

1 min 1 st time
1 min 2 nd timeX3
30 min 3 rd time

Fill up withbuffer
275 µL75 µL

0h Basal
sampleAspirate

25 µL

Fill up with
Fill up withworking dilution buffer
2h Basal
sample25 µL

2h Apical
sample25 µL

Incubate 2 h at
37⁰ C, 5% CO2

Cell culture
mediumAssay buffer

Assay conditions
Assay volumes•Apical: 75 µl
2Membrane area: 0.14 cm•Basal: 250 µl
Replicates: 3

Concentration: 10 µM

Time points: 2 (0 and 2 hours)

Sample volume: 25 µl

Working dilution

Sample for analysis

Quality control parameters
TEER: >100 Ω cm2
Lucifer Yellow•Paracellular flux: 2%•Papp : ≤ 4.5 x 10 -6 cm/s

Figure 5: General procedure for B to A assays.

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PreadyPort™ BCRP 96-well – Corning plate - User’s Manual

Evaluation of the Test Compound TransportThis section contains recommendations for sample retrieval, handling and evaluation.
For test compounds with unknown behavior, the following pattern for sample retrieval isrecommended:•

Donor side (apical for apical to basolateral, basolateral for basolateral to apical): t=0
and t =2 h•

Receptor side (basolateral for apical to basolateral, apical for basolateral to apical): t=0
and t=2 h

Shorter or longer periods may be required for very high or low permeability compounds.
If the experiment is performed using a specific BCRP inhibitor, this inhibitor must be incubatedin both compartments (donor and receptor sides) during the whole experiment.
For calculations the following formula is recommended:

PAPP =dQ1

dt A  C0

Where:•••

dQ/dt: amount of translocated material over incubation time (nmol/s)
2
A: area of insert (0.14 cm for PreadyPort BCRP-96)
C0: initial concentration of product applied in apical (A-B) or basal (B-A) compartment(nmol/ml)

The efflux Ratio (ER=Papp(B-A)/Papp(A-B)) is a general measure of the involvement of active
processes. ER values higher than 2 indicates an active process.
Considerations for calculations:•

Papp is traditionally given in units of 10-6 cm/s. The dimensions of all parameters must
be set accordingly (Q in mol, t in seconds, A in cm2, C0 in mol per cm3)•

When plotting Q versus time, the amount of material lost with previous samples mustbe accounted for•When applying the formula with a single time point that time point must be in thelinear range otherwise the obtained Papp will underestimate the real value. It is notrecommended to sample compounds with unknown behavior at a single time point

Normal Range of Controls (According to FDA, p. 64 Fig. A1)•••

The efflux ratio of prazosin must be greater than 2 in BCRP overexpressing MDCKII
cells
In the presence of Ko134 (specific BCRP inhibitor) the efflux ratio has to significantly
decrease > 50%
Paracellular flux of Lucifer Yellow must be less than 2% per hour of incubation (internal
data)

15
©READYCELL & SOLVO Biotechnology – Last version in October 2020 This product is manufactured in compliance with ISO9001:2015