Materials and Reagents

Notes:

1. Material and reagents are stored as per the manufacturer’s recommendation. 
2. Fluorescent peptides and glycosphingolipid-peptide fusions are used as described in Garcia-Castillo et al., 2018. The assay requires the presence of both a fluorophore and a biotin covalently attached to compound you want to measure (see Figure 3). Peptide fusions containing biotin and fluorophore attached to ganglioside GM1 were synthesized in-house using specialized methods described in (Garcia-Castillo et al., 2018). Precursor peptides were synthesized by New England Peptide (Gardner, MA, USA), and gangliosides were purchased from Dr. Sandro Sonnino (U. Milan, Italy). However, in theory, any macromolecule (peptide, protein, nucleic acid or chemical) containing both a fluorophore and biotin will work in this assay. An example compound is shown below. Here, lower-cased amino acids are depicting D-isomers. Since glycine (shown as upper-cased G) does not have chirality, the peptide essentially contains no L-amino acids that can be susceptible to degradation in vivo.
   
   
   Figure 3. Structure of peptide-lipid fusion constructs used in transcytosis assay 
   
   

1. Pipette tips (USA Scientific, catalog numbers: 1126-7810 , 1120-8810 , 1121-3810 )
2. 0.4 μm pore size Transwell^® inserts (Corning, catalog number: 3413 )
   Note: Transwell^® plates are kept at room temperature.
3. 96-well Assay Plate (No Lid, Black Flat Bottom, Non-treated, polystyrene) (Corning, Costar^®, catalog number: 3916 )
4. Aluminium-foil (Fisher Scientific, Fisherbrand^TM, catalog number: S05356A )
5. MDCK-II cells (Parental line from American Type Culture Collection) (ATCC, catalog number: CCL-34 ) (Kind gift from Dr. Steven Claypool)
6. Fatty acid-free bovine serum albumin (Sigma-Aldrich, catalog number: A6003-10G ) (lyophilized powder, essentially fatty acid free)
7. Pierce^TM Streptavidin magnetic beads (Thermo Fisher Scientific, catalog number: 88817 )
   Note: Beads are stored at 4 °C per the manufacturer's recommendation.
8. Formamide (Sigma-Aldrich, catalog number: F9037-100ML )
9. Dimethylformamide (DMF) (Sigma-Aldrich, catalog number: 227056 )
10. Biotin (Sigma-Aldrich, catalog number: B4501-1G )
11. DMEM 4.5 g/L D-glucose (Corning, catalog number: 10-013-CVR )
12. Pen/Strep, 100x (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140148 )
13. Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: A3160502 )
14. Tris base (Sigma-Aldrich, catalog number: T1503-1KG )
15. NaCl (Sigma-Aldrich, catalog number: S9625-10KG )
16. Tween 20 (Sigma-Aldrich, catalog number: P2287-500ML )
17. 0.25% Trypsin-EDTA (Thermo Fisher Scientific, Gibco^TM, catalog number: 25200072 )
18. PBS (Thermo Fisher Scientific, Gibco^TM)
19. EDTA (Sigma-Aldrich, catalog number: E4884-500G )
20. MDCK-II complete growth media (see Recipes)
21. Basolateral solution (see Recipes)
22. Apical solution (see Recipes)
23. TBS 10x (see Recipes)
24. 1x TBS-T (see Recipes)
25. 0.5 M EDTA pH 8.0 (see Recipes)
26. Elution buffer (see Recipes)
    

Equipment

1. Portable Pipet-Aid^® XP Pipette Controller (Drummond Scientific, model: 4-000-101 )
2. Incubator Forma Scientific CO₂ water-jacketed incubator (Thermo Fisher Scientific, model: Forma^TM Series II , catalog number: 3110)
3. Centrifuge (Eppendorf, model: 5415C )
4. EVOM2 Epithelial Voltohmmeter (World Precision Instruments, model: EVOM2 )
5. TECAN Spark multimode microplate reader (Tecan Trading, model: Spark^® )
6. MagneSphere^® Technology Magnetic Separation Stand (Promega, catalog number: Z5342 )
7. Sonicator (Stainless Steel AquaSonic Ultrasonic cleaner, VWR, model: 50T )
   

Software

1. GraphPad Prism 7
   GraphPad Software 7825 Fay Avenue, Suite 230 La Jolla, CA 92037 USA (https://www.graphpad.com/scientific-software/prism/)
   

Procedure

1. Day 1: Plating and polarizing cells
   
   1. Trypsinization
      
      1. Aspirate MDCK-II growth media from the stock cell culture flask used for passaging. 
      2. For a T-75 flask of MDCK-II cells, add 5-10 ml of warmed, sterile PBS. Gently tilt/rotate flask to wash the cells. Remove PBS.
      3. For a T-75 flask, add 3 ml of 0.25% Trypsin-EDTA. Tilt/rotate flask to make sure cell surface is evenly coated.
      4. Return to a 37 °C incubator with 5% CO₂ under humidified conditions for 5-10 min.
      5. Observe under a light microscope with phase contrast. If cells are properly detached, they will appear round and floating in suspension.
      6. Collect cell suspension in conical tube and pipet cells 6-8 times up and down. No clumps should be observed. Cell suspension should be a homogeneous mixture. 
   2. Add MDCK-II cells at a density of 200,000 cells in 200 μl in complete growth media (see Recipes) to the apical side of a Transwell^® insert (12-well 0.4 μm pore size Transwell^®-65 mm). In addition, 1 ml complete growth media is added to the basolateral chamber.
      
      1. Two Transwell^® inserts are prepared for (Untreated) controls, 2 Transwell^® inserts for reporter peptide, and 2 Transwell^® per glycosphingolipid-peptide fusion tested. 
      2. Each condition is tested as biological duplicates (i.e., 2 Transwell^® inserts per condition). Experiments must always include untreated and reporter-peptide controls.
   3. Incubate the plate for 2-3 days to allow for polarization at 37 °C with 5% CO₂ under humidified conditions.
   
   
2. Day 3: Preparation of testing glycosphingolipid compounds
   
   1. Centrifuge the tube containing lyophilized compound at 13,000 rpm (~13,800 x g) for 3 min to pellet the material to the bottom of tube.
   2. Add 1 part volume of DMF (actual volume depends on stock concentration–for our compounds, this is ~60 μl), sonicate for 30-60 sec, followed by ~5 sec vortex.
      Notes: 
      
      1. Sonication is done in a bath sonicator (VWR, AquaSonic, model 50T ).
      2. You want to reach a high final concentration in the ~50-200 μM range (in a tube containing approximately 100 μg of lyophilized peptide [MW ~2,103], add 50 μl of DMF and 100 μl of water to obtain a stock solution in the ~50-200 μM range). The concentration of all test compounds is determined using NanoDrop (For the Alexa Flour 488 used in our studies, we use absorbance at 495 nm).
      3. The solution may appear cloudy and orange-red at this point.
   3. Add 2 parts H₂O (for example 120 μl water to give a final volume of 180 μl) followed by a quick 5 sec vortex to reach a final 33% DMF in H₂O.
      Notes: 
      
      1. The solution should be bright green and clear of particulates. If it is orange, then add either more DMF or H₂O until it turns green. (The color change we observed is for an Alexa Flour 488 attached to the reporter peptide) 
      2. At this point, this stock solution will be used to make the final dilution into “Apical” media for adding onto cells.
   4. To prepare this Apical test solution, dilute the appropriate volume of stock into Apical Solution (see Recipes) to reach a final concentration of 0.1 μM.
      Note: The goal is to have a final 1:1 ratio of lipid to dfBSA.
   
   
3. Transcytosis in MDCKII cells
   
   1. Check electrical resistance of MDCK-II Transwell^® inserts after 2-3 days using EVOM Epithelial Voltohmmeter to measure integrity of tight junctions.
      Note: Acceptable electrical resistances of MDCK-II monolayers used for transcytosis experiments is in the 250Ω-300Ω range.
   2. Prepare apical and basolateral solutions (see Recipes).
   3. Wash transwells 2 times using serum-free DMEM. Replace media with apical (200 μl) and basolateral (1 ml) solutions in the respective chamber. 
   4. Allow cells to equilibrate for 15 min in a 37 °C/5% CO₂ cell culture incubator.
   5. Replace apical chamber with 200 μl apical solution containing 0.1 μM reporter peptide or 0.1 μM glycosphingolipid fusion.
      Note: Reporter peptide and glycosphingolipid fusion stocks range from 30 μM to 150 μM in 33% DMF/66% H₂O. 
   6. Incubate for 3 h in a 37 °C/5% CO₂ cell culture incubator.
   7. To quantify transcytosed peptide and lipid-peptide fusions, collect basolateral media in pre-labeled Eppendorf tubes and proceed to streptavidin pull-down assay. 
   8. To calculate the apparent permeability coefficient (PAPP, cm/sec), apical chamber solution is also collected in pre-labeled Eppendorf tubes. A standard curve ranging from 0 nM to 200 nM is used to interpolate concentration of peptide or lipid-peptide fusion in apical chamber after 3 h.
   
   
4. Streptavidin Pull-down Assay of basolateral media samples
   
   1. Wash Streptavidin magnetic beads 3 times with TBS-T.
      Notes:
      
      1. Beads are never vortexed. They are brought into solution in stock tube by gentle inverting.
      2. Ten microliter beads are needed per sample.
      Example: For 12 samples, place 120 μl Streptavidin beads in an Eppendorf tube and wash 3 times each with 1 ml TBS-T using magnetic rack.
   2. Resuspend 10 μl beads in 50 μl TBS then add to 1 ml basolateral sample
      Example: For 12 samples, resuspend 120 μl Streptavidin beads by adding 600 μl TBS. Then, add 50 μl Streptavidin beads to each basolateral sample.
   3. Incubate basolateral samples with streptavidin beads overnight at 4 °C with head-over rotation covered in foil.
   
   
5. Day 4: Elution and read-out of basolateral samples
   
   1. Collect the beads using a magnetic rack and wash 3 times with TBS-T.
      Note: Each wash is done with 1 ml TBS-T and beads mixed by inverting.
   2. Bound peptide or lipid-peptide fusions are eluted from beads by addition of 220 μl elution buffer (See Recipes) and boiling.
      Notes:
      
      1. Invert the beads to ensure that they are in solution.
      2. After beads are in solution, boil for 2 min at 65 °C in a heat block.
   3. After boiling, collect the beads using a magnetic rack. 
   4. Pipet 100 μl of each sample x 2 (technical replicates) on a black 96-well plate.
   5. Fluorescence is read on a TECAN Spark microplate reader for Alexa-488 channel and against a standard curve for each compound. 
      
      1. Settings:
         
         
      2. Sample standard curves (Figure 4)
         
         
         Figure 4. Sample standard curves for peptide (blue) and lipid-peptide fusion (red) used to calculate the amount of transcytosed compound. Plotted are the mean ± SEM of 3 technical replicates.
         
         
      3. A basolateral standard curve for each compound being tested is made in elution buffer ranging from 0 pM to 1,000 pM.
         
         1. Make 1 μM solutions of peptide and lipid-peptide stocks in elution buffer.
         2. Make 1 ml of 10 nM solutions (10 μl 1 μM + 990 μl elution buffer).
         3. Make serial dilutions with elution buffer beginning with 1,000 pM:
            1,000 pM = 100 μl 10 nM + 900 μl elution buffer
            500 pM = 500 μl 1,000 pM + 500 μl elution buffer
            250 pM = 500 μl 500 pM + 500 μl elution buffer
            125 pM = 500 μl 250 pM + 500 μl elution buffer
            62.5 pM = 500 μl 125 pM + 500 μl elution buffer
            31.5 pM = 500 μl 62.5 pM + 500 μl elution buffer
            15.6 pM = 500 μl 31.5 pM + 500 μl elution buffer
            0 pM = elution buffer

Data analysis

GraphPad Prism software is used to calculate the concentration of lipid-peptide fusion. As detailed above, a standard curve for each test compound is used to relate fluorescence intensity to a known concentration of peptide or lipid-peptide fusion. Absorbance at 495 nm is initially used to calculate stock concentrations. For detailed instructions on graphing and interpolating standard curves, the reader is referred to the GraphPad software website.
To calculate the apparent permeability coefficient (PAPP, cm/sec) a standard curve ranging from 0 nM to 200 nM is used to interpolate concentration of peptide or lipid-peptide solution remaining in the apical chamber after a 3 h continuous incubation.

The apparent permeability coefficient (PAPP, cm/sec) is calculated across cell monolayers grown in transwells based on the appearance rate of lipid-peptide fusions in the basolateral compartment over time:
Papp (cm/sec) = [VD/(A x MD)] x (DMR/Dt)
Papp (cm/sec) = [cm³/(cm² x mol)] x (mol/sec)

VD = apical (donor) volume (cm³) (e.g., 0.2 ml = 0.2 cm³)
MD = apical (donor) amount (mol)
A = membrane surface area (cm²) of apical (donor) chamber (i.e., transwell surface area = 0.33 cm² in this protocol)
DMR/Dt = the amount of compound (mol) transferred to the basolateral (receiver) compartment over time (sec).

Notes

1. This protocol is also used routinely in our lab with human intestinal T84 cells and human colonic Caco-2 cells.
2. Tight junction integrity must be confirmed using EVOM measurements.
3. To dissolve reporter peptide and glycosphingolipid-reporter peptide fusions first add DMF and sonicate for 30-60 sec, then add water.
4. Assay is done in serum-free DMEM. 
5. The molar ratio 1:1 of compound to defatted-BSA is used.
6. When preparing compound solutions, keep solutions at 37 °C and never place diluted lipids on ice as they may form micelles. 
7. A typical control for paracellular leak is a 4 °C temperature block to stop endocytosis/transcytosis machinery.
8. We use this assay in our lab to test the effect of gene silencing (using esiRNAs) and small molecule drugs (as is described for Dyngo-4a in Garcia-Castillo et al., 2018). 
9. Using this assay, we performed structure-function studies of glycosphingolipid-peptide fusions containing modifications to the ceramide moiety. Also, using this assay, we tested for transcytosis of the incretin hormone GLP-1 (glucagon-like peptide 1).
   

Recipes

1. MDCK-II complete growth media
   DMEM
   10% FBS
   1x Pen/Strep
   Store at 4°C
2. Basolateral solution (40 ml)
   40 ml DMEM
   400 mg defatted-BSA (df-BSA) (final concentration: 1% [w/v])
   Prepared fresh on the day of assay
3. Apical solution (40 ml)
   40 ml DMEM
   24.6 μl basolateral solution (Recipe 2, df-BSA final concentration: 0.1 μM)
   Prepared fresh on the day of assay
4. TBS 10x (1 L)
   24 g Tris base
   88 g NaCl
   Dissolve in 900 ml distilled water and pH 7.4
   Add distilled water to a final volume of 1 L
   Store at room temperature
5. 1x TBS-T (1 L)
   100 ml 10x TBS
   1 ml Tween 20
   900 ml distilled water
   Store at room temperature
6. 0.5 M EDTA pH 8.0 (1 L)
   186.1 g Na₂EDTA to 800 ml MilliQ H₂O
   Add NaOH to reach pH 8.0
   Add MilliQ H₂O up to 1 L
7. Elution buffer (95% Formamide, 10 mM EDTA, 0.4 mg/ml biotin) (100 ml)
   95 ml 100% formamide
   2 ml 0.5 M EDTA, pH 8.0
   40 mg biotin
   3 ml MilliQ H₂O
   

Acknowledgments

This project was supported by an NIH F-32 DK111072-01 to M.D.G-C; DK084424, DK048106, DK104868, and an unrestricted innovator grant from Novo Nordisk to W.I.L; DK104868 to D.C. We acknowledge the Harvard Digestive Disease Center DK034854. We are grateful to all members of the Lencer and Chinnapen laboratories for their helpful discussions.

Competing interests

The authors declare that there are no conflicts of interest or competing interests.

References

1. Fung, K. Y. Y., Fairn, G. D. and Lee, W. L. (2018). Transcellular vesicular transport in epithelial and endothelial cells: Challenges and opportunities. Traffic 19(1): 5-18. 
2. Garcia-Castillo, M. D., Chinnapen, D. J. and Lencer, W. I. (2017). Membrane transport across polarized epithelia. Cold Spring Harb Perspect Biol 9(9): a027912. 
3. Garcia-Castillo, M. D., Chinnapen, D. J., Te Welscher, Y. M., Gonzalez, R. J., Softic, S., Pacheco, M., Mrsny, R. J., Kahn, C. R., von Andrian, U. H., Lau, J., Pentelute, B. L. and Lencer, W. I. (2018). Mucosal absorption of therapeutic peptides by harnessing the endogenous sorting of glycosphingolipids. Elife 7: e34469.
4. Nelms, B., Dalomba, N. F. and Lencer, W. (2017). A targeted RNAi screen identifies factors affecting diverse stages of receptor-mediated transcytosis. J Cell Biol 216(2): 511-525. 
5. Thuenauer, R., Muller, S. K. and Romer, W. (2017). Pathways of protein and lipid receptor-mediated transcytosis in drug delivery. Expert Opin Drug Deliv 14(3): 341-351.