Immunofluorescence Detection and F-actin Staining of MTLn3 Cells   

Abstract

Materials and Reagents

1. MTLn3 cells
   
2. Type I rat tail collagen (BD Biosciences, catalog number: 354236 )
   
3. Leibovitz's L15 media w/o phenol red (Life Technologies, Invitrogen^TM, catalog number: 21083-027 )
   
4. Leibovitz's L15 media w/ phenol red (Life Technologies, Invitrogen^TM, catalog number: 11415-064 )
   
5. Rhodamin phalloidin red Cy3 (Life Technologies, Invitrogen^TM, catalog number: R415 )
   
6. Phosphate buffered saline (PBS)
   
7. Trypsin-EDTA
   
8. HCI
   
9. 95% ethanol
   
10. Acetic acid
    
11. FBS-alpha-MEM
    
12. Paraformaldehyde
    
13. Triton X-100
    
14. Glycine
    
15. BSA
    
16. N-propyl gallate
    
17. Glycerol
    
18. Methanol
    
19. Nail polish
    
20. Collagen solution
    
21. EGF (EMD Millipore, catalog number: 01-102 ) (see Recipes)
    
22. Rhodamine phalloidin (see Recipes)
    

Equipment

1. Centrifuges
   
2. Coverslips
   
3. Tissue culture flask
   
4. Parafilm
   
5. Culture dish
   
6. Humidified chamber
   
7. Sharp forceps
   
8. Water bath
   
9. Culture hood
   

Procedure

1. Collagen coated coverslip preparation:
   
   1. Wash coverslips twice with 95% ethanol.
      
   2. Wash and incubate the coverslips in IN HCI for 1 h at room temperature (RT).
      
   3. Wash twice with sterilized PBS (coverslips can be stored in methanol in bulk).
      
   4. If taken straight from methanol, wash twice with sterilized PBS to rehydrate the coverslips. From this point on every procedure needs to be conducted under sterile conditions.
      
   5. Monolayer the coverslips on 100 mm culture dish.
      
   6. Coat the coverslips in acetic acid-collagen solution:
      256 μl of 3.33 m ml^-1 type I collagen into 20 ml of filter sterilize 0.01 M acetic acid. Add 20 ml of acetic acid collagen solution to the monolayer coverslips in 100 mm dish. Incubate in the culture hood at RT for 2 h.
      
   7. After collagen coating, rinse once gently with sterilized PBS. At this point the coverslips can be stored in PBS at 4 °C for 1 week.
   
   
2. Splitting MTLn3 cells on collagen coated coverslips and starvation:
   
   8. For 80 mm tissue culture flask, MTLn3 cells should be kept 50-70% confluent at all times. Over confluent cells do not respond to EGF stimulation well.
      
   9. Rinse once with 1 ml trypsin-EDTA, aspirate and then trypsinize with 1 ml trypsin-EDTA at 37 °C incubator for 3-5 min until cells come off the flask.
      
   10. Add 9 ml 5% FBS-alpha-MEM into the flask. Pipet the cells up and down a few times to break the cell clumps.
       
   11. From a 70% confluent 80 mm flask, take 3-5 ml cells to one 100 mm culture dish of the collagen coated coverslips. Usually it takes > 24 h for MTLn3 cells to be well spread on the coverslips.
       
   12. Wash once with warm starvation media, 0.35% BSA-L15 media, and starve the cells in this media for 3 h at 37 °C without CO₂.
       
   
   
3. EGF stimulation and cell fixation:
   
   13. Place culture plate into 37 °C water bath. Add appropriate volume of EGF stimulation buffer to the plate and stimulated for 3 min.
       
   14. Fix cell with warm 4% Paraformaldehyde in 37 °C water bath for 10 min.
       
   15. After fixation, rinse 1x with PBS and the coverslips can be stored in PBS at 4 °C overnight.
   
   
4. Immunofluorescence/ F-actin staining
   
   16. Permeablize the fixed cells with 0.5% triton X-100-PBS for 10 min at RT.
       
   17. Optional: rinse once with 0.1 M glycine-PBS and incubate in 0.1 M glycine-PBS for 10 min at RT to reduce background.
       
   18. Block with 1% FBS- 1% BSA- PBS at RT 4x 5 min, then transfer coverslips onto parafilm.
       
   19. Incubate 1-2 h with primary antibody (50 μl per coverslips) usually start with 1:200 dilution in 1% BSA-PBS buffer in a humidified chamber.
       
   20. Wash with PBS or 1% BSA-PBS 5 min 3-5 times to wash out non-specific primary antibody.
       
   21. Incubate with 1: 200 secondary antibody in 1% BSA-PBS for 1 h at RT.
       
   22. Aspirate the secondary antibody, and incubate 20 min with Rhodamine phalloidin (1:15 dilution in 1% BSA-PBS)
       
   23. Wash the coverslips with PBS 5 times for 5 min each.
   
   
5. Mounting coverslips:
   
   24. Making mounting buffer (0.11 g n-propyl gallate + 2.5 ml 2x PBS + 2.5 ml glycerol), vortex until all of it has dissolved (usually takes 40 min). Store in dark for 2 weeks.
       
   25. Add 3 μl mounting solution on the slide. Use sharp forceps to pick up the coverslip, absorb excess buffer on the edge of the coverslips, carefully drop the coverslip on mounting solution.
       
   26. Apply nail polish on four corners of the coverslip and seal the edge with nail polish all round. Store the slides either in -20 °C or 4 °C in slide boxes.

Recipes

1. To make EGF aliquots
   Spin down the EGF powder, add 333 μl PBS and then aliquot into either 5 μl or 10 μl aliquots. Store in -80 °C.
   
2. To make Rhodamine phalloidin stock
   Spin down the power and add 1.5 ml methonol to dissolve. Store in -20 °C in the dark.
   

References

1. Capani, F., Deerinck, T. J., Ellisman, M. H., Bushong, E., Bobik, M. and Martone, M. E. (2001). Phalloidin-eosin followed by photo-oxidation: a novel method for localizing F-actin at the light and electron microscopic levels. J Histochem Cytochem 49(11): 1351-1361.
   
2. Yip, S. C., El-Sibai, M., Coniglio, S. J., Mouneimne, G., Eddy, R. J., Drees, B. E., Neilsen, P. O., Goswami, S., Symons, M., Condeelis, J. S. and Backer, J. M. (2007). The distinct roles of Ras and Rac in PI 3-kinase-dependent protrusion during EGF-stimulated cell migration. J Cell Sci 120(Pt 17): 3138-3146.