Permeabilized Epithelial Cells to Study Exocytic Membrane Transport

I. INTRODUCTION
Polarized exocytic transport in epithelial cells can be measured by performing in vitro assays on filter-grown Madin-Darby canine kidney cells (MDCK strain II cells). Techniques have been developed to establish conditions where cytosol-dependent transfer of a viral marker protein is monitored in MDCK cells permeabilized with the cholesterol-binding and pore-forming toxin Streptococcus streptolysin-O (SLO). The transport is assayed either early in the biosynthetic pathway, from the endoplasmic reticulum (ER) to the Golgi complex, or from the trans-Golgi network (TGN) to the apical or from the TGN to the basolateral plasma membrane (for a detailed characterization of the assays and their properties, see Lafont et al., 1998).

The assays provide the possibility to analyze the effects of exogeneous molecules added to the cytosol. Cytosolic molecules can be inactivated prior to their addition to permeabilized cells or depleted using antibodies. Membrane-impermeable molecules can gain access to lipids or membrane proteins facing the cytosol. We have successfully used fluorescent lipid analogues, chemicals, antibodies, peptides, purified proteins, and toxins. The specificity of the inhibition of transport after depleting cells for a selected cytosolic molecule may be tested by rescuing transport when the purified molecule is added back to the transport reaction. Efficiency of the transport steps can also be tested after cells have been treated with drugs affecting biosynthetic pathways, e.g., after cholesterol biosynthesis inhibition using methyl-β-cyclodextrin.

II. MATERIALS AND INSTRUMENTATION
The recombinant SLO fused to a maltose-binding protein is obtained from Dr. S. Bhakdi (University of Mainz, Mainz, Germany). PhosphorImager and ImageQuant software are purchased from Molecular Dynamics. Sonifier cell disruptor B 15 is purchased from Branson. Media and reagents for cell culture are purchased from Gibco Biocult and Biochrom.; chemicals are from Merck; ATP (Cat. No. A-6033), antipain (Cat. No. A-6271), CaCO₃ (Cat. No. C-5273), chymostatin (Cat. No. C-7268), cytochalasin D (Cat. No. C- 5394), leupeptin (Cat. No. L-2884), pepstatin (Cat. No. P-4265), and pyruvate (Cat. No. P2256) are from Sigma; creatine kinase (Cat, No. 127566), creatine phosphate (Cat. No. 621714), dimathyl sulfoxide (DMSO) (Cat. No. D-5879), endoglycosidase H (Cat. No. 1088734), and NADH (Cat No. 107727) are from Boehringer Mannheim; trypsin and soybean trypsin inhibitor are from Worthington Biochemical Corporation; protein A-Sepharose (Cat. No. 17-0780-01) is from Pharmacia; and cell filters (0.4-µm pore size; No. 3401, 12-mm-diameter Transwell polycarbonate filters) are from Costar.

III. PROCEDURES A. SLO Standardization
Each batch of toxin is standardized for the amount of lactate dehydrogenase (LDH) released from the filter-grown MDCK cells, and the amount of LDH released is determined according to a previously described protocol. Alternatively, it is possible to use antibody cytosol accessibility to monitor SLO permeabilization.

Solutions

1. Growth medium (GM): Eagle's minimal essential medium with Earle's salts (E-MEM) supplemented with 10mM HEPES, pH 7.3, 10% (v/v) fetal calf serum, 2mM glutamine, 100U/ml penicillin, and 100µg/ml streptomycin.
2. Either purified SLO or recombinant SLO fused to a maltose-binding protein is obtained from Dr. S. Bhakdi (University of Mainz, Mainz, Germany). The recombinant SLO preparation is strored as lyophilisate at -80°C.
3. 10X KOAc transport buffer: To prepare 1 liter, dissolve 250mM HEPES (59.6 g); 1150 mM KOAc (112.9 g), and 50mM MgCl₂ (25ml from 1M stock) in 800ml water. Adjust the pH to 7.4 with-50ml 1M KOH and make it 1 liter with water.
4. KOAc+ buffer: KOAC buffer containing 0.9mM CaCO₃ (90mg/liter) and 0.5mM MgCl₂ (0.5ml/liter from 1M stock)
5. Assay buffer: KOAc plus 0.011% Triton X-100 for media and KOAc for filter
6. NADH 14 mg/ml KOAc
7. 60mM pyruvate: 30mg in 50ml water

Steps

1. Grow MDCK cells strain II on 1.2-mm-diameter filters for 3.5 days in vitro and change the medium every 24h.
2. Wash cells by dipping in KOAc.
3. Prepare a range of SLO, e.g., from 0.5 to 10 µg of recombinant SLO per filter. Activate the SLO for 30 min at 37°C in 50 µl KOAc plus 10mM dithiothreitol (DTT) per filter. Place the filters on a Parafilm sheet put on a metal plate on an ice bucket.
4. Add 50µl of activated SLO on the apical side for 15min at 4°C.
5. Wash excess SLO by dipping the filters twice in KOAc+.
6. Transfer the filters to a 12-well dish containing 0.75 ml TM at 19.5°C. Add 0.75 ml TM to the apical side and incubate at 19.5°C for exactly 30min in a water bath.
7. Collect apical and basolateral media, cut the filters, and shake the filters with 1 ml KOAc containing 0.1% Triton X-100.
8. Mix in a disposable cuvette:
   1. 800 µl of assay buffer (KOAc or KOAc/Triton x-100)
   2. 200 µl of sample
   3. 10 µl of NADH
   4. 10 µl of pyruvate.
   
   
   Turn the cuvette upside down twice using Parafilm to close it.
9. Read immediately the OD at 340nm for 60 s (the OD should be around 1.5).
10. Calculate the LDH activity as the change in OD in 10s, taking the average of the first 30s.
11. Dissolve the recombinant SLO in KOAc buffer and store in aliquots at -80°C with the amount necessary for one set of assays per aliquot.

B. Preparation of Cytosols
Solutions

1. Growth medium: For HeLa cells, use Joklik's medium supplemented with 50ml/liter newborn calf serum (heat inactivated for 30min at 56°C), 2 mM glutamine, 100U/ml penicillin, 100µg/ml streptomycin, and 150 µl 10 M NaOH. For MDCK cells, use Eagle's minimal essential medium with Earle's salts (E-MEM) supplemented with 10mM HEPES, pH 7.3, 10% (v/v) fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.
2. 10X KOAc transport buffer: To prepare 1 liter, dissolve 250mM HEPES (59.6 g); 1150 mM KOAc (112.9 g), and 50mM MgCl₂ (25ml from 1M stock) in 800ml water. Adjust the pH to 7.4 with -50ml 1M KOH and make it 1 liter with water.
3. PBS+: PBS containing 0.9mM CaCl₂ and 0.5 mM MgCl₂.
4. Swelling buffer (SB): For 100ml, prepare 1 mM EGTA 0.5M (200µl), 1 mM MgCl₂ 1M (100µl), 1 mM DTT 1M (100 µl), and 1 µM cytochalasin D 1 mM (100 µl).
5. Protease inhibitor cocktail (CLAP): To prepare a 1000X stock, dissolve antipain, chymostatin, leupeptin, and pepstatin each at 25µg/ml DMSO and combine.

Steps

HeLa Cytosol

1. To grow 20 liters of HeLa cells in suspension to a density of 6 × 10⁵ cells/ml:
   1. Inoculate 250ml cell suspension (6 × 10⁵ cells/ml) in 1 liter medium and leave stirring in a 37°C room.
   2. On the third day, split 1:4 by adding 250ml to 750ml of fresh medium. Leave stirring in the 37°C room. This will give 4 liters of cell suspension.
   3. On the fifth day, again split 1:4 by adding 1 liter of the cell suspension to 3 liters of fresh medium in a 6-liter round-bottom flask. If the cells seem overgrown, add some fresh medium. Thus, between 16 and 20 liters of cell suspension are obtained.
2. Concentrate the cell suspension to 2 liters either by centrifugation or with any cell-concentrating system.
3. Centrifuge cells at 5000rpm in a Sorvall GS-3 rotor (400-ml buckets) at 4°C for 20min.
4. Discard the supernatant and wash cells by resuspending the pellet with 10ml of ice-cold PBS with a sterile 10-ml pipette (10 ml PBS/bucket). Pool the suspension in 250-ml Falcon tubes.
5. Spin at 3000rpm for 10min at 4°C in a minifuge. The pellet will be loose. Discard the supernatant.
6. Fill the tube with cold SB (about 35ml). Resuspend the cells carefully with a sterile 10-ml pipette in SB. Let the pellet swell for 5 min on ice.
7. Spin in the minifuge at 2000rpm at 4°C for 10 min. Remove as much as possible of the supernatant. The pellet should be loose.
8. Transfer the pellet to a 30-ml Dounce homogenizer (use a spatula) and, after adding SB, perform five strokes. Add 0.1 volume of 10× KOAc and homogenize further by 15-20 strokes.
9. Spin the homogenate at 4°C at 10,000rpm for 25 min in SS-34 tubes (Sorvall). Collect the supernatant.
10. Spin at 50,000 rpm in Ti70 tubes (Beckmann) for 90min at 4°C. Aliquot the supernatant in screw-top 1.5-ml tubes. Avoid including lipids that might be on the top of the supernatant. Freeze the aliquots in liquid N₂ and store at -70°C.
11. Measure the protein concentration; it should be around 5 mg/ml. Lower concentrations do not work.
12. When needed, thaw the aliquots quickly and keep them on ice (up to 6 h) until use. Aliquots can be refrozen at least twice.

MDCK Cytosol

1. Trypsinize thirty 24 x 24-cm dishes of confluent MDCK cells. Resuspend trypsinized cells in cold growth medium (containing 5% FCS to inactivate trypsin) and leave on ice until all dishes are trypsinized and cells are pooled. After this, all handling should be done on ice using ice-cold solutions.
2. Centrifugate at 4°C for 10 min at 2000 rpm in a RF Heraeus centrifuge. Wash medium away with PBS.
3. Wash in PBS containing 2mg/ml STI. Wash out STI with PBS and resuspend the cell pellet in the SB (cf. see earlier discussion) and keep 10min on ice.
4. Centrifugate at 2000 rpm for 10 min at 4°C in a RF Heraus centrifuge.
5. Sonicate the loose cell pellet (~10ml) (power 6, 0.5-s pulse, sonifier cell disruptor B 15, Branson) until cells are broken as judged by light microscopy. Add 0.1 volume of 10X KOAc to the sample and spin it for 20 min at 3000rpm.
6. Spin the supernatant again at 75,000rpm for 1h in a TLA 100.2 rotor.

The procedure routinely yields ~6ml of cytosol at 14mg/ml. Other cell types, e.g., NIH 3T3 fibroblasts, can also be used as starting material for cytosol preparation using the protocol just described. We have not observed significant differences between the efficiencies of the different cytosols to support transport. However, HeLa cytosol is used routinely because of the ease of preparing large quantities.

C. Transport Assays
Solutions
All solutions are sterilized and kept at 4°C unless indicated.

1. Growth medium: E-MEM supplemented with 10mm HEPES (10ml/liter), pH 7.3, 10% (v/v) fetal calf serum, 2mM glutamine 200mm (10ml/liter), 100U/ml penicillin 10⁴U/ml (10ml/liter), and 100 µg/ml streptomycin 10⁴µg/ml (10ml/liter).
2. Infection medium (IM): E-MEM supplemented with 10mm HEPES, pH 7.3, 0.2% (w/v) BSA, 100 U/ml penicillin, and 100µg/ml streptomycin.
3. Labelling medium (LM): Methionine-free E-MEM containing 0.35g/liter sodium bicarbonate instead of the usual 2.2 g/liter 10mm HEPES, pH 7.3, 0.2% (w/v) BSA supplemented with 16.5µCi of [³⁵S]methionine/ filter.
4. Chase medium (CM): Labelling medium without [³⁵S]methionine and containing 20µg/ml cycloheximide and 150µg/ml cold methionine.
5. 10X KOAc transport buffer: To prepare 1 liter, dissolve 59.6g HEPES (250 mM); 112.9 g KOAc (1150mM), and add 25 ml from 1M stock MgCl₂ (50 mM) in 800ml water. Adjust the pH to 7.4 with ~50 ml 1M KOH and make it 1 liter with water.
6. KOAc+ buffer: KOAC buffer containing 0.9mM CaCO₃ (90mg/liter) and 0.5mm MgCl₂ (0.5ml/liter from 1M stock)
7. PBS+: PBS containing 0.9mM CaCl₂ (0.13g/liter CaCl₂·2H₂O) and 0.5mM MgCl₂ (0.5ml/liter from 1M stock).
8. 0.5M EGTA: To prepare 100 ml, dissolve 19 g in 60ml water. The solution should be turbid (pH 3.5). Add slowly ~10 ml 10M KOH. When the pH of the solution starts to clear, adjust to 7.4. Make it 100ml with water.
9. 0.1 M Ca and 0.5M EGTA: To prepare 100 ml, stir 1 g CaCO₃ and 3.8 g EGTA in -70ml water for at least 45 min (degas). After adding 2 ml of 10 M KOH, the pH is about 6. Finally, make the pH 7.4 adding few drops of 1M KOH. Make it 1 liter with water.
10. Transport medium (TM): To prepare 50ml, combine 50µl 1M DTT, 200µl 0.5M EGTA, and 1 ml 0.5M EGTA/0.1M CaCO₃ in 1X KOAc.
11. ATP-regenerating system (ARS): 100X stock, prepare three solutions (10ml each):
    1. 100mM ATP (disodium salt, pH 6-7, neutralized with 2M NaOH; 0.605g/10ml)
    2. 800mM creatine phosphate (disodium salt, 2.620 g/10ml)
    3. 800U/mg (at 37°C) creatine kinase (0.5 mg/10 ml in 50% glycerol).
       Store stocks in aliquots at -20°C. Mix solutions a-c 1:1:1 just before use.
12. Lysis buffer (LB): PBS+ containing 2% NP-40 and 0.2% SDS.
13. CLAP: To prepare a 1000X stock, combine 25µg/ ml DMSO of antipain, chymostatin, leupeptin, and pepstatin.

Steps
The basic steps of the assays can be summarized as follows. Grow MDCK cells on a permeable filter support until a tight monolayer is formed. Grow cells on 12-mm-diameter filters and use when they display a transmonolayer electrical resistance of at least 50Ω× cm². Infect cells layers with either vesicular stomatitis (VSV) or influenza virus using the G glycoprotein of VSV (VSV G) or the hemagglutinin (HA) of influenza virus N as basolateral or apical markers, respectively. After a short pulse of radioactive methionine, block the newly synthesized viral proteins either in the ER at 4°C or in the TGN after a 20°C incubation. At this stage, one cell surface is permeabilized with SLO, which allows leakage of cytosolic proteins, whereas membrane constituents, including transport vesicles, are retained inside the cells. After removal of the endogenous cytosol by washing, add cytosol and ATP and raise the temperature to 37°C. Transport of the viral proteins from the ER to the Golgi complex or from the TGN to the apical or the basolateral plasma membrane is reconstituted in a cytosol-, energy-, and temperature-dependent manner. Measure the amount of viral proteins reaching the acceptor compartment by endoglycosidase H treatment (ER to Golgi transport), trypsinization of HA on the apical surface (TGN to apical transport), or immunoprecipitation of the VSV G at the basolateral surface (TGN to basolateral transport). Obtain quantitations of viral polypeptides resolved on SDS-PAGE by PhosphorImager analysis.

Due to the handling and the various steps, the entire procedure for running one assay requires about 9 h for the apical assay and 11 h for the basolateral assay. Carry out immunoprecipitation for the basolateral assay the following day (takes 5h). The ER-to-Golgi transport takes about 6h before an overnight enzymatic treatment step. Samples can be frozen at -20°C before running SDS-PAGEs (routinely performed the following day). It is worth noting that for the reproducibility of the results the assays should be performed strictly, obeying the schedule indicated in the protocol.

Grow the N strain influenza virus (A/chick/ Germany/49/Hav2Neq1) in 11-day embryonated chick eggs as described. Prepare a stock of phenotypically mixed VSV (Indiana strain) grown in Chinese hamster ovary C15.CF1 cells, which express HA on their plasma membrane as described. Prepare the affinity-purified antibody raised against the luminal domain of the VSV G as described.

Apical Transport Assay

1. Cell culture: Seed 1:72 of the MDCK cells from one confluent 75-cm² flask per filter (6 × 10⁴ cells/filter) with 0.75 ml of growth medium apically and 2 ml basolaterally. Change the growth medium every 24h and use cells 3.5 days after plating.
2. Infection: Prior to viral infection, wash the monolayers in warm PBS+ and then IM and infect with the influenza virus in 50µl IM (20pfu/cell; enough to obtain 100% infection as judged by immunofluorescence) on the apical side. After allowing adsorption of the virus to the cells for 1 h at 37°C, remove the inoculum and continue the infection for an additional 3h after adding 0.75 ml IM on the apical side and 2ml IM on the basolateral side of the filter.
3. Metabolic labeling: Rinse the cell monolayers by dipping in beakers containing warm PBS+ and LM at 37°C. Place a drop of 25µl of the LM containing 12.5 µCi of [³⁵S]methionine on a Parafilm sheet in a wet chamber at 37°C in a water bath. Add 100µl of only LM to the apical side of the filters and place them basal side down on the drop. Incubate for 6 min at 37°C.
4. TGN block: Terminate all the pulse by moving the filters to a new 12-well plate containing 1.5ml CM already at 20°C. Add 0.75 ml of CM (20°C) on the apical side and incubate for 75 min in a 19.5°C water bath.
5. SLO permeabilization: Activate the SLO in KOAc buffer containing 10mM DTT at 37°C for 30min and keep at 4°C until used for permeabilization. Use the toxin within 1 h of activation. Wash the filters twice in ice-cold KOAc+ by dipping. Carefully remove excess buffer from the apical side and blot the basolateral side with a Kleenex. Place 25-µl drops of the activated SLO (enough to release 60% LDH in 30min, about 20mg/ml) on a Parafilm sheet placed on a metal plate on an ice bucket. Leave the apical side without buffer. Place the filters on the drop for 15 min. Wash the basolateral surface twice by dipping in ice-cold KOAc+ buffer. Transfer the filters to a 12-well dish containing 0.75 ml TM at 19.5°C. Add 0.75 ml TM to the apical side and incubate at 19.5°C for exactly 30min in a water bath.
6. Transport: Remove the filters from the water bath, rinse them once with cold TM, blot the basolateral side, and place on a 35-µl drop on either TM (control) or HeLa cytosol (±treatment or molecule to be tested) supplemented each with ARS (3 µl/100 µl TM or HeLa cytosol). Add the ARS to TM or cytosol immediately before dispensing the drops onto which the filters are placed. Layer 100 µl of TM on the apical side and incubate at 4°C for 15 min in a moist chamber. Transfer the chamber to a warm water bath for 60min at 37°C. Terminate the transport by transferring on ice and washing the filters with cold PBS+ three times.
7. Trypsinization: Add to the apical surface 250µl of 100µg/ml trypsin freshly prepared in PBS+ and then add 2 ml of PBS+ to the basolateral chamber. Keep on ice for 30min and stop the reaction by adding 3 µl of soybean trypsin inhibitor (STI; 10mg/ml) to the apical side and then wash the apical surface three times with PBS+ containing 100µg/ml STI.
8. Cell lysis: Solubilize the monolayers in 100 µl LB containing freshly added CLAP. Scrape the cells and spin for 5min in a microfuge; discard the pellet. Analyze 20 µl per sample by SDS-PAGE on a 10% acrylamide gel, fix, and dry the gel.
9. Quantitation: Scan the gels with a PhophorImager and measure the band intensities using ImageQuant software. Calculate the amount of HA transported as the percentage of HA (68 kDa) transported to the cell surface = [2 x HA2/(HA + 2 x HA2) x 100] with HA2 (32kDA) being the small trypsin cleavage product of HA.

Basolateral Transport Assay

1. Cell culture: Seed 1:72 of MDCK cells from one confluent 75-cm² flask per filter (6 × 10⁴ cells/filter) with 0.75 ml of growth medium apically and 2 ml basolaterally. Change the growth medium every 24h and use cells 3.5 days after plating.
2. Infection: Prior to viral infection, wash the monolayers in warm PBS+ and then in IM and infect with the vesicular stomatitis virus in 50µl IM (20pfu/cell; enough to obtain 100% infection as judged by immunofluorescence) on the apical side. After allowing adsorption of the virus to the cells for 1 h at 37°C, remove the inoculum and continue the infection for an additional 3h after adding 0.75ml IM on the apical side and 2ml IM on the basolateral side of the filter.
3. Metabolic labeling and chase: Rinse the cell monolayers by dipping in beakers containing warm PBS+ and then LM at 37°C. Place a drop of 25µl of the LM containing 12.5BCi of [³⁵S]methionine on a Parafilm sheet in a wet chamber at 37°C in a water bath. Add 100µl of LM to the apical side of the filter and place the filter on the drop. Incubate for 6 min at 37°C and incubate for an additional 6min at 37°C in CM with 0.75 ml on the apical side and 2ml on the basolateral side before the 19.5°C block.
4. TGN block: Terminate the pulse by moving the filters to a new 12-well plate containing 1.5ml CM already at 20°C. Add 0.75 ml of CM (20°C) on the apical side and incubate for 60min in a 19.5°C water bath.
5. SLO permeabilization: Activate the SLO in KOAc buffer containing 10mM DTT at 37°C for 30min and keep at 4°C until used for permeabilization. Use the toxin within 1 h of activation. Wash the filters twice in ice-cold KOAc+ by dipping. Carefully remove excess buffer from the apical side and blot the basolateral side with a Kleenex. Add 50-µl drops of the activated SLO (enough to release 60% LDH in 30min, about 40mg/ ml) on a Parafilm sheet placed on a metal plate layered on an ice bucket. Leave the basolateral side without buffer. Incubate for 15min. Wash the apical surface twice with 0.75 ml of ice-cold KOAc+ buffer. Transfer the filters to a 12-well dish containing 0.75ml TM at 19.5°C. Add 0.75ml TM to the apical side and incubate at 19.5°C for exactly 30min in a water bath.
6. Transport: Remove the filters from the water bath, rinse them once with cold TM, blot the basolateral side, and add 100µl of either TM (control) or HeLa cytosol (±treatment or molecule to be tested) supplemented with ARS each (3 µl/100 µl TM or HeLa cytosol) on the apical side. Add the ARS to TM or cytosol prior to dispension onto drops. Incubate at 4°C for 15 min. Put the filters on already dispensed 35-µl drops of TM supplemented with ARS on a Parafilm in a moist chamber in a water bath at 37°C. Incubate for 60 min. Terminate the transport by transferring on ice and washing the filters with cold PBS+ three times.
7. Anti-VSV G binding: Wash twice (2-5min) with 2 ml of CM containing 10% FCS (CM-FCS) on the basolateral side and once with 0.75 ml on the apical side. Dilute anti-VSV-G antibodies in CM-FCS (1:18, i.e., 50Bg/ml; 300 µl) and place the filters on 25-µl drops of PBS+/ab (1.5µl ab/filter). Add nothing on the apical side and incubate in a cold room on a metal plate placed onto an ice bucket for 90 min. Remove the filters and wash (three times) on the basolateral side with CM-FCS (2ml with constant rocking) and once on the apical side. Shake the filters gently for efficient removal of unbound antibodies. Wash the filters once with PBS+ and place each on a 25-µl drop of PBS+ supplemented with cold virus (1:25; 300µl). Incubate for 10 min in the cold.
8. Cell lysis: Solubilize the monolayers in 200µl LB containing freshly added CLAP and cold virus (1:166). Scrape the cells, spin for 5 min, and discard the pellet. Remove a 10-µl aliquot from each sample (total).
9. Immunoprecipitation: Wash protein A-Sepharose powder with PBS (3×), let it swell 10 min in PBS, wash it with LB, and store as 1:1 slurry in LB at 4°C for 3 weeks maximum. Add 30 µl of the 1 : 1 slurry of protein A-Sepharose to the lysate. Rotate in a cold room for 60 min. Spin the resin down and wash (3×) with 500 µlLB, elute the bound sample with 35 µl 2X Laemmli buffer, and boil for 5 min at 95°C. Load 20µl of bound material and 10µl of total (after boiling with 10µl of 2X Laemmli buffer) on SDS-PAGE gels (10%). Run SDS-PAGE on 10% acrylamide gels, fix, and dry the gels.
10. Quantitation: Scan the gels with a Phophor- Imager and measure band intensities using Image- Quant software. Calculate the amount of VSV G (67 kDa) transported as follows: percentage of VSV G on the cell surface = (surface immunoprecipitated VSV G / total VSV G) x 100.

ER-to-Golgi Transport Assay
In the ER-to-Golgi transport assay, influenza N- or VSV- infected MDCK monolayers can be used. An infection with the influenza virus is used here as an example.

1. Cell culture: Seed 1:72 of the MDCK cells from one confluent 75-cm² flask per filter with 0.75ml of growth medium apically and 2ml basolaterally. Change the growth medium every 24h and use cells 3.5 days after plating.
2. Infection: Prior to viral infection, wash the monolayers in warm IM and then infect with the influenza virus in 50 µl IM (20 pfu/cell; enough to obtain 100% infection as judged by immunofluorescence) on the apical side. After allowing adsorption of the virus to the cells for 1 h at 37°C, remove the inoculum and continue the infection for an additional 3 h after adding 0.75 ml IM on the apical side and 2ml IM on the basolateral side of the filter.
3. Metabolic labeling: Rinse the cell monolayers by dipping in beakers containing warm PBS+ and then LM at 37°C. Place a drop of 25 µl of the LM containing 12.5 BCi of [³⁵S]methionine on a Parafilm sheet in a wet chamber at 37°C in a water bath. Add 100µl of LM to the apical side of the filter and place the filter on the drop. Incubate for 6 min at 37°C.
4. Terminate the pulse by moving the filters to a new 12-well plate containing 1.5 ml CM already at 4°C. Add 0.75 ml of CM (4°C) on the apical side and incubate for 30min at 4°C.
5. SLO permeabilization: Activate the SLO in KOAc buffer containing 10mM DTT at 37°C for 30min and keep at 4°C until used for permeabilization. Use the toxin within 1 h of activation. Wash the filters twice in ice-cold KOAc+ by dipping. Carefully remove excess buffer from the apical side and blot the basolateral side with a Kleenex. Place 25-µl drops of the activated SLO (enough to release 60% LDH in 30min, about 20mg/ml) on a Parafilm sheet placed on a metal plate layered on an ice bucket. Leave the apical side without buffer. Place the filters on the drops for 15 min. Wash the basolateral surface twice by dipping in ice-cold KOAc+ buffer. Transfer the filters to a 12-well dish containing 0.75 ml TM at 4°C. Add 0.75 ml TM to the apical side and incubate at 37°C for 3 min and 1.5 ml on the basolateral side of the filter (formation of pores), followed by an incubation at 4°C for 20min in fresh TM on both sides (cytosol depletion).
6. Transport: Rinse the filters once with cold TM, blot on the basolateral side, and put on a 35-µl drop of either TM (control) or HeLa cytosol (±treatment or molecule to be tested) supplemented with ARS each (3µl/100µlTM or HeLa cytosol). Add the ARS to TM or cytosol prior to dispension onto drops. Layer the apical side with 100µl of TM and incubate at 4°C for 15 min in a moist chamber. Transfer the chamber to a water bath at 37°C for 45 min. Terminate the transport by transferring on ice and washing the filters with cold PBS+ three times.
7. Cell lysis: Solubilize the monolayers in 100µl LB containing freshly added CLAP. Scrape the cells and spin for 5 min in microfuge; discard the pellet.
8. Endoglycosidase H treatment: Remove a 75-µl aliquot and add to 25 µl of 0.2M sodium citrate buffer, pH of 5.0. The resulting 100 µl mixture has a pH of 5.3. Divide it in two 50-µl aliquots. One receives 5µl of 1 U/ml endoglycosidase H and the other receives only the citrate buffer. After 20 h at 37°C, terminate the reaction by boiling in Laemmli buffer. Analyze the samples by running SDS-PAGE on a 10% acrylamide gel, fix, and dry the gel.
9. Quantitation: Scan the gels with a PhophorImager and measure band intensities using ImageQuant software. Calculate the amount of HA transported as the percentage of HA acquiring endoglycosidase H resistance with the following formula: percentage of HA reaching the Golgi complex = (endo H-resistant HA / total HA) x 100.

IV. COMMENTS AND PITFALLS
In all cases the values are expressed as control cytosol-dependent transport being 100% (transport in the presence of cytosol minus transport in the absence of added cytosol). For each manipulation a matched control is used (e.g., antibody or peptide tested vs. control antibody or peptide, respectively). Assays are carried out routinely on duplicate filters and quantifications represent the mean ± SEM obtained in several experiments.

A critical parameter for the successful performance of these transport assays is the quality of SLO. MDCK cells are difficult to permeabilize compared to several other cell types (e.g., BHK, CHO, and L cells), and with the available commercial sources of SLO, the degree of permeabilization, as measured by LDH release, has not been satisfactory. The wild-type toxin purified from Streptococci and the recombinant toxin produced in Escherichia coli have both worked equally well.

The available amount of reagent often determines whether the preincubation during cytosol depletion is possible, as cytosol depletion must be carried out in an excess volume of buffer. The routinely used volume (750 µl per filter) can be, however, somewhat reduced. By using 500µl per filter there is not yet a significant effect on transport efficiency, and by using 200µl per filter cytosol depletion is compromised moderately, which increases the background, cytosol-independent transport, and results in a transport efficiency of about three-fourths of normal.

Because the transport is carried out in a leaky cellular microenvironment with diluted cytosolic components, the increase in transport obtained with exogenous cytosol is usually two- to threefold (threeto fourfold in the ER-to-Golgi assay). This is the window in which the differences in cytosol dependent transport are measured. In assays measuring transport in the late secretory pathway, part of the efficiency is lost due to retention of some viral marker early in the exocytic route. However, because both markers, HA and VSV G, are glycoproteins whose mobility on SDS-PAGE shifts according to the degree of glycosylation, careful examination of their mobilities will reveal, in apical and basolateral assays, if the test condition retarded significantly the processing of the marker to the terminally glycosylated form. This may therefore serve as an internal control for the specificity of inhibition. A more accurate way to test the effect of a reagent in the early secretory pathway is to assay the ER-to-Golgi transport. The real advantage of having established similar procedures for three different transport assays is the possibility of using them as internal controls for each other. This enables the identification of molecules that are specifically involved in either apical or basolateral transport routes and allows the discrimination between compounds that are needed only in the polarized routes versus those that are common to all three transport processes.

Reference
Lafone, E, Verkade, P., and Simons, K. (1998). Annexin XIIIb associates with lipid microdomains to function in apical delivery. J. Cell Biol. 142, 1413.