96-Well Plates (Inserts) with Seeded and Differentiated (21days) Caco-2 Cells

USER’S MANUAL (Corning Plate)CacoReady™ 96 User’s Manual-Corning Plate

TABLE OF CONTENTS
I. CacoReady™ USE-ASSAY FLOW CHART ...................................................................................... 2
II. SOLUTIONS & EQUIPMENT ....................................................................................................... 3
1. Solutions .............................................................................................................................. 3
2. Equipment ............................................................................................................................. 3
III. Unpacking and Maintenance ................................................................................................... 4
1. Unpacking ............................................................................................................................ 4
2. Replacing the Shipping Medium.......................................................................................... 4
3. Exchanging the Caco-2 Cell Culture Medium ........................................................................ 5
IV. QUALITY CONTROL OF THE BARRIER SYSTEM ......................................................................... 6
1. TEER Measurement ............................................................................................................. 6
2. Lucifer Yellow Paracellular Permeability Assay ................................................................... 7
V. APPENDIX I: GENERAL PROTOCOL FOR PERMEABILITY ASSAYS ............................................... 8
1. General Protocol for Apical-To-Basal Permeability Studies ................................................ 8
2. General Protocol for Basal-To-Apical Permeability Studies .............................................. 11
3. Evaluation of the Compound Permeability. ...................................................................... 13
4. Reference Compounds for Permeability Studies .............................................................. 14

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CacoReady™ 96 User’s Manual-Corning Plate

I. CacoReady™ USE-ASSAY FLOW CHART
Receiving Day

Assay Performance Day

Shipping on Tuesday*See note below

Replace the Shipping medium on
FridayRecommended day to perform permeability
assays: Monday

Figure 1. Assay Flow Chart*Note: Assays can be performed from Monday to Friday of the week following receipt of theplates. Monday is the recommended day (equivalent to 21 days of barrier differentiation) for
measuring TEER values and starting experiments. If this schedule is followed, no extra medium
exchanges are required. Medium exchanges are required if the permeability experiments are
performed on Tuesday through Thursday. Please consult Table 1 below, for guidelines on
exchanging the medium.

DAY OF EXPERIMENTMEDIUM EXCHANGE

MondayTuesday

MondayWednesday

MondayThursdayMonday and Wednesday

FridayMonday and Wednesday

Table 1. Assay and Medium Exchange Guidelines.

** IMPORTANT NOTE: Due to our previous experience with long-distance shipments and/or
extreme temperatures at a destination, we strongly recommend to measure the TEER on
Monday and in case TEER values are low, perform a medium change and wait until
Wednesday to let the cells recover. On Wednesday, read the TEER again and perform theassay accordingly.

helptech@readycell.com
reagents@readycell.com+ 34 93 403 70 77

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CacoReady™ 96 User’s Manual-Corning Plate

II. SOLUTIONS & EQUIPMENT
Note: The reagents listed below are not provided with the kit and must be made and available
for use prior to delivery of CacoReady™ plates.

1. Solutions
i. Standard Caco-2 Cell Culture Medium••••

DMEM 1g/L glucose (REF. BE12-707F – LONZA)
10% FBS (REF. S181B-500 – BIOWEST)
1% Glutamine 200 Mm (REF. BE17-605F – LONZA)
1% Penecillin (10,000 U/ml) – Streptomycin (10/mg/ml) (REF. DE17-602F – LONZA)
Note: If you would like to use other references, please check that the characteristics of the
product are similar. Please feel free to contact us with any queries or doubts.

ii. HBSS (10X) Buffer••••

54.00 mM KCl
4.40 mM KH2PO4
1.37 M NaCl
3.30 mM Na2HPO4Adjust to pH ~ 7.4, autoclave and store at 4°C until use.
iii. CaCl2·2H2O (100mM), autoclave and store at 4°C until use.
iv. MgCl2·6H2O (50mM), autoclave and store at 4°C until use.
v. Ethanol (70%)
vi. Lucifer Yellow – (REF. L0259 – SIGMA)
vii. The HBSS (1X) + Ca2+/Mg2+ Buffer should be prepared from the above stock solution(item ii, above) on the day of experiments as follows:
For 25 ml:

2.5 ml HBSS (10X) +0.55 ml MgCl2·6H2O (50 mM) +0.325 ml CaCl2·2H2O (100 mM) +Adjust final volume with 21.625 ml of sterile MilliQ water.

Note 1: For transport studies of more than 2 hours, D-glucose should be added (at final
concentration: 5mM) to the working HBSS buffer.
Alternatively, commercial HBSS Transport Buffer can be purchased from SIGMA (REF: H8264).
Note 2: If necessary, apical HBSS pH can be adjusted to 6.5 with 1N HCl.

2. Equipmenti.

Cell culture incubator, 37°C, in a highly humidified atmosphere of 95% air and 5% CO2

ii.

Sterile cell culture cabinetiii.

CO2 gas tankiv.

Reservoir plates (Provided by ReadyCell™). Please note that the reservoir should besterilized and kept sterile for further use.
3

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CacoReady™ 96 User’s Manual-Corning Plate
Note: If you are interested in using the receiver plate 96-well (not provided by ReadyCellTM) thereference is 3382 – CORNING.

v.

Water bath, 37°C

vi.

Aspiration System. It is highly recommended to use the following manifold for
aspiration: REF. 3-000-093 – DRUMMOND

vii.

Automatic multichannel micropipettor (volume range: 50-1200 µl).

viii.

Sterile culture medium containers (REF. 4870 – CORNING).

ix.

TEER measurement equipment: REF. EVOM2 – WPIx.

Fluorimeter, HPLC-UV/MS, liquid scintillation counter, etc.

Note: We highly recommend the use of WPI electrode ref. STX100C96 (This element is notprovided with the TEER measurement equipment and should be purchased separately).

III. Unpacking and Maintenance
1. Unpacking
Upon receipt of the CacoReady product, retrieve the zip-top bags containing the plates. Openthe zip, and leave the bag at a dark location at room temperature until Friday (refer to Table 1,
above).

2. Replacing the Shipping Medium
Caution: Never handle more than one plate at a time while replacing the shipping medium.
Re-solidification of the shipping medium may provoke mechanical damage to the cellmonolayer.
i. Retrieve the plates from the bags and remove the Parafilm wrap.
ii. Incubate the plates in a 5% CO2 humidified atmosphere at 37°C for 4 hours.
iii. Remove the CacoReady™ plate (one at the time, if you have multiple plates) from the
incubator and place inside the laminar flow hood, along with one reservoir plate.
iv. Using sterile procedures (inside the laminar flow hood):- Fill a sterile reagent reservoir with 50 ml of pre-warmed (37°C) Caco-2 culture
medium.
- Open the CacoReady™ plate and the reservoir plate, and leave the lids upwards, next
to the plate.
- Carefully lift the apical inserts of the CacoReady™ plate and transfer onto the reservoir
plate.
- Remove all liquefied shipping medium from the wells in the basal compartments of the
CacoReady™ plate via aspiration with the 96-well manifold.
- Using a multichannel pipette, dispense 235 µl of the Caco-2 cell culture medium fromthe sterile reservoir into the wells of the basal compartment (column by column) of
the CacoReady™ plate.
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CacoReady™ 96 User’s Manual-Corning Plate- Using the aspiration manifold connected to a vacuum (adjust aspiration flux tomedium-low), aspirate the liquefied shipping medium from the apical inserts of the
CacoReady™ plate placing the manifold perpendicular to the cell monolayer and close
to the insert wall. Check that the shipping medium has been removed from all wells.
10 µl of medium will be left in each well.
- Using a multichannel pipette, dispense 75 µl of the Caco-2 cell culture medium fromthe sterile reservoir, and fill, column by column, each of the apical inserts of the
CacoReady™. Always add the medium against the wall of the well, not onto the center
directly on the cell monolayer.
- Carefully return the apical inserts onto the basal compartment of the CacoReady™
plate. Replace the lid and place inside cell culture incubator, set at 37°C, with 5% CO2
until Monday.
v. Once the shipping medium has been replaced by the fresh Caco-2 cell culture medium,
plates should be kept inside the incubator until the following Monday (Day 21).

CacoReady ™ plate

Reservoir plate

CacoReady ™ plate

Reservoir plate
Remove liquefied
shipping medium

1
5

Add fresh Caco-2
medium2
Remove liquefied
shipping medium

63
Add fresh Caco-2
medium7

4

Shipping
mediumCaco-2
mediumFigure 2. Scheme for Replacing the Shipping Medium

IMPORTANT NOTE: Keep the reservoir plate provided by ReadyCell™. It will be used during thepermeability assay. Please note that the reservoir should be sterilized and kept sterile forfurther use.

3. Exchanging the Caco-2 Cell Culture Medium
Exchanging the cell culture medium has to be carried out once (at day 21) or twice (at day 21
and day 23) depending on the day of the assay (See table 1, above), following the sameprocedure described in (Section III-2, page 4).

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CacoReady™ 96 User’s Manual-Corning Plate

IV. QUALITY CONTROL OF THE BARRIER SYSTEM
1. Pre-assay Quality Control: TEER Measurement
This section provides general instructions for TEER measurements. It is important to carefully
read the instructions of your TEER measurement equipment in conjunction with the directions
listed below:
On Monday (equivalent to day 21 of cell culture), perform TEER measurement on theplate/plates before any further processing.
To proceed with the TEER measurement, follow the steps below:
i. It is highly recommended to use the WPI STX 100C96 electrodes, in order to avoid any
damage to the cell monolayer.
ii. Sterilize the electrodes (probe) by submerging both electrodes in ethanol (70%) for 30
minutes.
iii. Equilibrate the electrodes (probe) for 5 minutes in Caco-2 cell culture medium, pre-warmed
at room temperature.
iv. At the same time, remove the 96-Insert HTS plate with Caco-2 cells from the incubator(Section III-iii, page 4), place in a bio-safety cabinet, and allow it to reach to room
temperature (approximately 20 minutes), as TEER measurements need to be performed atroom temperature.
v. Place the electrode on top of the apical insert and proceed with themeasurement.
vi. A TEER value of 500 Ω·cm2 (or higher) indicates that the barrier system is
acceptable for an absorption assay (Active membrane area is 0.14 cm2).

http://www.wpiinc.com

KEY DATA
Membrane area
Minimum TEER value

0.14 cm2
500 Ω·cm2

IMPORTANT NOTES••••TEER reading should be carried out 48 hours after exchanging medium, in order toverify the integrity of the monolayer prior to the permeability assay. Never performthe TEER measurement with the shipping medium.
Do not perform repeated TEER measurements within the same insert.
For an appropriate TEER measurement, both electrodes have to be well submerged
within the cell culture medium in both apical and basal compartments.
Please, see Important Note page 2

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CacoReady™ 96 User’s Manual-Corning Plate

2. Post-assay Quality Control: Lucifer Yellow Paracellular Permeability
Assay
Prepare a Lucifer Yellow (LY) Stock Solution of 1mg/ml in sterile ddH2O. Aliquot (For example 500µl) and store at –20°C. Pre-warm (37°C) the Working dilutions of LY before use (see Figure 3,
below for recommended Working dilutions).
To proceed with the LY permeability assay follow the steps below:i)

Following the procedure described in Section III-2, page 4., gently rinse both the apicaland the basal compartments of the 96-insert HTS Plate with HBSS Buffer Working
solution (75 µl apical / 225 µl basal).

ii) Add 75 µl of LY Working dilutions, as recommended in Figure 3 below, into the wellson the wells in the apical compartment.
iii) Add 225 µl of HBSS Buffer Working solution onto the basal compartment.
iv) Incubate the 96-Insert HTS plate protected from light in a 37°C incubator for 1 hour.
v) Recover 200 µl from each well in the basal compartment and dispense onto
corresponding wells on an empty flourometer plate, (mix well and avoid bubble
formation when dispensing each aliquot).
vi) Read the fluorescence intensity directly in a fluorometer, using a 485 nm excitation
filter and a 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 Lucifer Yellow Standard Curve

Note: Please note that under standard conditions (i.e., in HBSS Buffer), 21-day CacoReady™
barriers present LY Paracellular Flux values of ≤ 0.7% after 1 hour, and/or Papp values of ≤ 1 x
10-6 cm/s. These values are considered as reference values for the monolayer integrity.
KEY DATALY Paracellular Flux
LY Papp

0.7%≤ 1 x 10-6 cm/s

IMPORTANT NOTES:••

Perform the LY Assay after the permeability assay.
It is highly recommended to use some of the inserts only for testing LY as quality
control of the monolayer status.
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CacoReady™ 96 User’s Manual-Corning Plate

V. APPENDIX I: GENERAL PROTOCOL FOR PERMEABILITY ASSAYS
1.

General Protocol for Apical-To-Basal Permeability Studies

The protocol below describes a suggested procedural guide to perform A-B permeability
assays.
i.

Assay conditions••••

Test compound concentration: 10 µM (recommended)
Replicates:3
Time points: 2 (0 and 2 hours)
Assay volumes:• Apical: 75 µl of the test compound at working dilution (100 µl is added to theapical compartment and 25 µl is recovered at time 0hr.)•ii.

• Basal: 250 µl of HBSS Buffer Working solution
HBSS Buffer Working solution for 25 ml:• 2.5 ml HBSS (10X) +• 0.55 ml MgCl2, 6H2O (50 mM) +• 0.325 ml CaCl2, 2H2O (100 mM) +• 0.125 ml D-glucose (500 mM)
PROTOCOL (using a 96-well manifold for aspiration)

CAUTION: Do not add the Lucifer yellow reagent together with the test compounds. The
Lucifer Yellow could interfere with certain substances producing false results.
1- Prepare a 10 mM stock solution of the test compounds, using the proper solvent.
2- Prepare 10 µM working dilutions of test compounds in HBSS Buffer Working solution (seeabove, Section V-1-i).
3- Fill a sterile container for cell culture medium with 50 ml of pre-warmed (37°C) HBSS Buffer
Working solution.
4- Unwrap one reservoir plate under the biosafety cabinet.
5- Take 1 CacoReady™ plate from the incubator and place it in the biosafety cabinet next to
the reservoir plate.
6- Gently lift the apical inserts of the CacoReady™ plate and transfer them on top of thereservoir plate.
7- Remove all Caco-2 medium from the wells on the basal compartment of the CacoReady™
plate by aspiration with the 96-well manifold. Place the manifold perpendicular to the cellmonolayer and close to the insert wall.
8- Fill, column by column, each of the 96 wells of the basal compartments of the CacoReady™
plate with 250 µl of pre-warmed HBSS Buffer Working solution.
9- Remove the Caco-2 medium from the apical inserts of the CacoReady™ plate by aspiration
with the manifold. Place the manifold perpendicular to the cell monolayer and close to theinsert wall.
10-Fill, column by column, each of the 96 apical inserts of the CacoReady™ plate with 75 µl of
pre-warmed HBSS Buffer Working solution.
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CacoReady™ 96 User’s Manual-Corning Plate
11-Return the apical inserts onto the wells of the basal compartment of the CacoReady™ plate(original position).
12-Keep the plate for minimum of 1 min at room temperature inside the biosafety cabinet.
13-Repeat rinsing the plate (Steps 8 and 9, of Section V-1, ii, above) two more times and keep
the plate for 30 min at 37° C inside a 5% CO2 incubator.
14-Take the plate from the incubator, return it to the biosafety cabinet and place it next to thereservoir plate.
15-Gently lift the apical inserts of the CacoReady™ plate and transfer them to the top of thereservoir plate.
16-Remove the buffer from the wells in the basal compartments of the CacoReady™ plate by
aspiration with the 96-well manifold.
17- Fill, column by column, each of the 96 wells of the basal compartment of the CacoReady™
plate with 250 µl of pre-warmed HBSS Buffer Working solution.
18-Remove the buffer from the apical inserts of the CacoReady™ plate by aspiration with the
manifold.
19-Fill, column by column, each of the 96 apical inserts with 100 µl of the test compound
working dilution.
20-Recover 25 µl from the apical inserts (0 hr. Apical samples) and keep them at -20°C until
analysis.
21-Return the apical inserts onto the wells of the basal compartment of the CacoReady™ plate(original position).
22- Cover the CacoReady™ plate with its lid, and place it inside the 5% CO2 incubator, at 37°C,
for 2 hours.
23-Take the plate from the incubator, return it to the biosafety cabinet and place it next to thereservoir plate. Transfer the apical inserts onto the reservoir plate.
24-Recover 25 µl from the apical inserts (2 hr. Apical samples), and keep them at -20°C until
analysis.
25- Recover samples of 25 µl from the wells of the basal compartment (2 hr. Basal samples),and keep them at -20°C until analysis.
26- Proceed to sample analysis (0 h and 2 h samples, above).
27- After quantification of the test compound, proceed to calculate permeability coefficient(Papp) as indicated in “Evaluation of the Compound Permeability” Section V-3, below.

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CacoReady™ 96 User’s Manual-Corning Plate

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
Membrane area: 0.14 cm2•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: >500 Ω cm2
Lucifer Yellow•Paracellular flux: 0,7%
•Papp : ≤ 1 x 10 -6 cm/s

Figure 4. Diagram Showing the Steps involved in the Apical to Basal Permeability Assay Protocol

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CacoReady™ 96 User’s Manual-Corning Plate

2.

General Protocol for Basal-To-Apical Permeability Studies

The B-A permeability assay protocol remains essentially the same as the A-B protocol
described above, except for the fact that the test compound is added to the basal
compartment, acting as a donor side this time (See Figure 5). In this case, 275 µl of the test
compound is added to the basal compartment and 25 µl is recovered at time 0 (0 hr. basal
sample) while 75 µl of the HBSS Buffer Working solution is added to the apical compartment.
All other steps are identical as in the AB protocol, above.
••••

Test compound concentration: 10 µM (recommended)
Replicates: 3
Time points: 2 (0 and 2 hours)
Assay volumes:• Apical: 75 µl of HBSS Buffer Working solution.
• Basal: 250 µl of the test compound at working dilution (275 µl is added to the•

HBSS Buffer Working solution for 25 ml:• 2.5 ml HBSS (10X) +• 0.55 ml MgCl2, 6H2O (50 mM) +• 0.325 ml CaCl2, 2H2O (100 mM) +• 0.125 ml D-glucose (500 mM)

apical compartment and 25 µl is recovered at time 0hr.)

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CacoReady™ 96 User’s Manual-Corning Plate

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: >500 Ω cm2
Lucifer Yellow•Paracellular flux: 0,7%
•Papp : ≤ 1 x 10 -6 cm/s

Figure 5. Diagram Showing Steps involved in the Basal to Apical Permeability Assay Protocol

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CacoReady™ 96 User’s Manual-Corning Plate

3.

Evaluation of the Compound Permeability.

PAPP =dQ1

dt A  C0

Where:
dQ/dt: amount of product present in basal (A-B) or apical (B-A) compartment as afunction of time (nmol/s).
A: area of transwell (cm2).
C0: initial concentration of product applied in apical (A-B) or basal (B-A) compartment(nmol/ml).

Based on in vitro/in vivo correlation studies (see Table 2, below), Papp values obtained from
Caco-2 barrier studies are used to define and predict oral/intestinal, brain bioavailability or
toxicity of the tested compounds.
In vitro Papp Values

Range of Predicted in vivo Absorption

Papp ≤ 10-6 cm/s

Low (0-20%)

10-6 cm/s < Papp ≤ 10 x 10-6 cm/s

Medium (20-70%)

Papp > 10 x 10-6 cm/s

High (70-100%)

Table 2. Prediction of ‘in vivo’ Oral Absorption in Humans Based on ‘in vitro’.
Papp Values Obtained from Caco-2 Barrier Studies

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CacoReady™ 96 User’s Manual-Corning Plate

4.

Reference Compounds for Permeability Studies

Different markers can be used to check if a Caco-2 barrier (21-day) is acceptable for transport
studies. Data related to experimental conditions and standard Papp values for different
markers are provided below, and should be considered by end users as guidelines to confirm
the CacoReady™ barrier quality and performance.

Figure 6. Papp Values for Reference Compounds

APICAL-BASAL
Reference
CompoundPermeability
Range

Concentration
(µM)

Low

BASAL-APICAL
Efflux Ratio

Papp(10-6 cm/s)

Standard
Deviation

Papp(10-6 cm/s)

Standard
Deviation

10.00

0,15

0,02

0,07

0,01

0.51

1.00

14,00

5,40

12,70

3,60

0,90

1.00

14,20

5,30

11,90

3,80

0,80

10.00

19,42

1,18

11,14

0,69

0.57

10.00

49,87

4,65

19,35

1,65

0,39

Diclofenac

10.00

57,59

6,44

48,52

9,40

0,84

Quinidine

10.00

10,00

2,01

35,69

6,36

3,57

10.00

1,57

0,71

34,14

8,18

21,79

10.00

11,66

3,22

38,86

14,84

3,13

Digoxine

10.00

0,54

0,47

8,06

2,32

14.80

Vinblastine

10.00

1,86

0,18

44,07

9,52

23,72

Atenolol
Pindolol
Pindolol+CsA

MediumMetoprolol
Propranolol

High

Ciclosporine
Loperamide

Pgp
substrate

Table 3. Experimental Conditions and Papp Values for Reference Compounds.

helptech@readycell.com
reagents@readycell.com+ 34 93 403 70 77
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