Protocols / Handling

SUBCULTURING

Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.

Remove and discard culture medium from sub-confluent cultures (70-80%). During routine subculture the cells should always be subcultured before they achieve confluence.

Briefly rinse the cell layer with 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.

Add 1 to 3 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).

Note: Cells can clump if not separated into a single cell suspension when split. To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.

Add 5 to 10 mL of complete growth medium and aspirate cells by gently pipetting.

Add appropriate aliquots of the cell suspension to new culture vessels.The recommended inoculum is 1 X 104 viable cells/cm2. Subculture cells when they are about 80% confluent, at a cell concentration between 8 x 104 and 1 x 105 cell/cm2.

Incubate cultures at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:6 is recommended.

Medium Renewal: 1 to 2 times per week.

Usual Subculturing Frequency: 1 time per week.


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APICAL TO BASAL PERMEABILITY ASSAY

General protocol for Apical-To-Basal permeability studies 

Drug candidates or lead compounds are applied to the apical side of the cell monolayer (upper compartment of the insert), and their apical-to-basal (A-B) transport through the cell barrier is evaluated by sample recovering and compound detection at the basal side (lower compartment) over a defined incubation time. A-B permeability of compounds is determined as the coefficient of apparent permeability (Papp) in cm/s.
The protocol provided below describes a suggested procedure guide to perform screening permeability assays. 

Default conditions:

• Concentrations: 10 µM 
• Replicates:3
• Time points: 2 (0 and 2 hours)

Volumes:

• Apical: 250 µl
• Basal: 750 µl


 
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Buffer 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) 

 

Note 1: HBSS should be pre-warmed up to 37°C to avoid temperature stress while you are performing the experiments on the Caco-2 monolayers.
Note 2: Do not add the Lucifer yellow reagent together with the test compounds. The Lucifer Yellow could interfere with certain substances producing false results.

 

- Prepare a 10 mM stock solution of test compounds using the proper solvent.

- Prepare 10 µM working dilutions of test compounds in HBSS (x1) + Ca2+/Mg2++ D glucose buffer.

- Fill a sterile container for cell culture medium with 50 ml of pre-warmed (37°C) HBSS (x1) + Ca2+/Mg2++ D glucose buffer.

- Unwrap one “receiver plate” (CORNING Ref. 3526), within the biosafety cabinet.

- Remove all Caco-2 medium from the basal compartments of the Transwell plate via aspiration using the 24-well manifold.

- Fill, column by column, each of the 24 basal compartments of the Transwell plate with 750 µl of pre-warmed (37°C) HBSS (x1) + Ca2+/Mg2++ D glucose buffer.

- Remove the Caco-2 medium of the apical compartments of the Transwell plate via aspiration with the manifold.

- Fill, column by column, each of the 24 apical compartments of the Transwell plate with 250 µl of pre-warmed (37°C) HBSS (x1) + Ca2+/Mg2++ D glucose buffer.

- Smoothly transfer the apical integrated inserts (compartments) of the Transwell plate onto the top of the basal compartments of the Transwell plate (original position).

- Keep the plate for 1 min at room temperature inside the biosafety cabinet.

- Repeat washing steps. 

- Repeat washing steps.

- Keep the plate for 30 min at 37° C and 5% CO2 inside the incubator.

- Smoothly lift up the 24-integrated apical (upper) compartments of the Transwell plate and transfer them on top of the “receiver plate”.

- Remove all HBSS (x1) + Ca2+/Mg2++ D glucose buffer from the basal compartments of the Transwell plate via aspiration using the 24-well manifold.

- Fill, column by column, each of the 24 basal compartments of the Transwell plate with 750 µl of pre-warmed (37°C) HBSS (x1) + Ca2+ /Mg2+ + D glucose buffer.

- Remove the HBSS (x1) + Ca2+/Mg2+ + D glucose buffer of the apical compartments of the Transwell plate via aspiration with the manifold.

- Fill, column by column, each of the 24 apical compartments of the Transwell plate with 275 µl of compound working dilution.

- Recover 25 µl from the apical compartments (Apical Time 0 h sample) and keep them at -20 °C until analysis.

- Smoothly transfer the apical integrated inserts (compartments) of the Transwell plate onto the top of the basal compartments of the Transwell plate (original position).

- Incubate for 2h.

- Take the plate from the incubator and place it back within the biosafety cabinet besides the receiver plate. Split apical compartments from basal compartments in order to stop permeability assay.

- Recover 25 µl from the apical compartments (Apical Time 2 h sample), and keep them at -20°C until analysis.

- Recover samples of 25 µl from the basal compartments (Time 2 h samples) in Eppendorff tubes, and keep them at -20°C until analysis.

- Proceed to sample analysis (Time 0 h and Time 2 h samples).

- After quantification of test compound within samples, proceed to calculate permeability coefficient (Papp) as described into the literature.

 

LUCIFER YELLOW PARACELLULAR PERMEABILITY ASSAY

Lucifer yellow-CH is regularly used as a marker for paracellular permeability, to determine Caco-2 monolayer integrity, though other compounds may be appropriate. Prepare a Lucifer Yellow stock solution of 1mg/ml in sterile ddH2O. Aliquot it and store it at –20°C. Pre-warm (37°C) the working dilutions of LY before application (see figure 3 for recommended working dilutions).  

a) Rinse gently both the apical and basal compartment with HBSS (300 µl apical / 900 µl basal).
b) Add 250 µl of LY 100 µM on the apical compartment.
c) Add 750 µl of HBSS buffer on the basal compartment.
d) Incubate the 24-Insert HTS plate protected from light in a 37°C incubator for 1 hour.
e) Recover 200 µl from the basal compartment and dispense them onto an empty fluorometer plate (homogenize and avoid bubble formation when aspirating).
f) Read directly in a fluorometer using a 485 nm excitation filter and an emission filter of 527 nm.

 

Imagen2

 

Paracellular flux (or permeability) represents a marker for the status of the intercellular junctions when polarized monolayers are formed. When correctly constituted as a polarized and selective barrier sealed by tight junctions, they present no or minimal paracellular flux, leaving the transcellular pathway as the only way to cross the monolayer. The use of molecules that are unable to cross transcellularly and the study of their flux and permeability values, allow to evaluate the barrier status of the polarized cell monolayers grown on top of microporous membranes from inserts or insert culture systems.