Isolation of Epithelial Cells from Mouse Gastrointestinal Tract for Western Blot or RNA Analysis    

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In this protocol

Original research article

A brief version of this protocol appeared in:
Oncogene
May 2012

 

Abstract

The gastrointestinal (GI) tract is lined by a single layer of epithelial cells which function in secretion, absorption, and digestion. In addition, most GI tract tumors develop from epithelial cells (carcinomas). This protocol describes isolation of the surface epithelium from the underlying stroma, muscular layer and submucosa in the GI tract. In this protocol, epithelial cell adhesions are weekend by chelating Ca +2 ions followed by mechanical separation of the cells by vortexing. Analysis of protein levels and gene expression patterns in isolated epithelial cells versus whole GI tissue minimizes the potential for confounding contributions from contaminating stromal cells.

Keywords: Intestine tissue, Colon tissue, Cell isolation, Protein, Mouse

Materials and Reagents

  1. 4% sodium hypochlorite (Sigma-Aldrich, catalog number: 239305 )
  2. Phosphate buffered saline (PBS) (Mediatech, Cellgro®, catalog number: 21-031 )
  3. KCl
  4. NaCl
  5. KH2PO4
  6. Na2HPO4
  7. EDTA
  8. DTT
  9. Aprotinin (USB, catalog number: 9087-70-1 )
  10. Leupeptin (MP, catalog number: 195623 )
  11. Pepstatin (MP, catalog number: 195368 )
  12. PMSF (Sigma-Aldrich, catalog number: 329-98-5 )
  13. Ethanol
  14. 0.04% sodium hypochlorite (see Recipes)
  15. 100x protease inhibitor (store at -20 °C) (see Recipes)
  16. Solution B (see Recipes)
  17. 1x lysis buffer (see Recipes)

Equipment

  1. CO2 chamber for mice
  2. Dissecting scissors (one medium size and one small size for dissecting mice)
  3. Forceps (2)
  4. 15-ml conical tubes
  5. 1.5-ml eppendorf tubes
  6. 3ml Insulin syringe
  7. Vortex
  8. Centrifuge compatible with 15-ml conical

Procedure

  1. Make sure to prepare all the solutions including PBS at least 1 h before the start of killing the mice and store them on ice so they are cold when you start to work with them.
  2. Sacrifice the mouse (18-25 grams) by CO2 asphyxiation according to American Veterinary Medical Association (AVMA) guidelines on Euthanasia, 2007 (https://www.avma.org/KB/Policies/Documents/euthanasia.pdf). Briefly, first carbon dioxide’s anesthetic properties are used by exposing rodents to a slowly rising concentration of carbon dioxide until they become unconscious. A chamber fill rate of 20%, or 1/5 th of the cage volume per minute is recommended. Slower, uncoordinated movements occur after about 1 min, animals stop moving after about 2 min, and unconsciousness occurs after about 2.5 min. After unconsciousness is achieved, flow can be increased to hasten death. Two-step euthanasia is used to ensure death. Once the animal has lost consciousness and is unresponsive to a toe pinch, a follow-up method is utilized. This can include decapitation with sharp scissors, cervical dislocation, or thoracotomy.
  3. Open the abdomen and dissect the gastrointestinal tract from the stomach to anus by pulling gently on the stomach and removal of the mesentry (the peritoneal folding around the intestine which connects the intestinal tract to the dorsal abdominal wall. It contains fat, blood vessels and lymph nodes).
  4. Cut the intestine into pieces of interest (Jejunum, Ileum and large intestine).
  5. Keep the pieces in cold PBS on ice and work with them one at a time.
  6. Flush the content of the intestine with cold PBS to a 3 m clean it using l insulin syringe. Alternatively, invert the intestine on a glass rod and swirl in cold PBS
  7. Wash intestinal pieces in cold PBS and keep them until you are done with all pieces (to synchronize your work).
  8. Incubate pieces in 50- 100 ml of 0.04% sodium hypochlorite on ice for 15 min (this works well using a small beaker). This step removes bacterial contaminants.
  9. During this time prepare three 15-ml conical tubes for every tissue piece and put 5-10 ml of solution B in each tube (if you are using glass rods. 10 ml will be necessary). Mark the tubes and leave them on ice.
  10. Remove intestinal pieces from the sodium hypochlorite and rinse in PBS.
  11. Put the intestinal pieces in the 15 ml conical containing solution B for 15 min on ice.
  12. Remove solution B and add 5 ml PBS (or solution B). I do this by holding the intestinal piece with a long forceps, decanting the solution B then adding back the intestinal piece and add PBS.
  13. Vortex for 15 sec.
  14. Take the intestinal pieces and put them in another 15 ml conical with solution B.
  15. Repeat steps 11-14 twice (you will have 3 tubes per piece).
  16. Suspend the cells from all tubes for every piece and mix them in one tube.
  17. Centrifuge at 1,000 rpm (1,000 x g, 10 min at 4 °C).
  18. Take the fluid off and suspend the cells in lysis buffer (5 ml for Jejunum and Ileum or 1 ml for colon) for total cell lysate or Trizol (Invitrogen) 1 ml for RNA extraction.

Recipes

  1. 0.04% sodium hypochlorite
    1 ml 4% sodium hypochlorite
    99 ml PBS
  2. Solution B
    2.7 mM KCl 0.2 g or 2.7 ml 1 M KCl
    150 mM NaCl 8.77 g 50 ml 3 M NaCl solution
    1.2 mM KH2PO4 0.16 g
    680 mM Na2HPO4 9.65 g
    1.5 mM EDTA 0.44 g 3 ml 0.5 M EDTA solution
    0.5 mM DTT 0.08 g 1 ml 0.5M DTT (add fresh)
    Bring to 1 L in ddH2O
  3. 100x protease inhibitor (store at -20 °C)
    10 mg aprotinin
    10 mg leupeptin
    10 mg pepstatin
    174.2 mg PMSF
    Bring to 10 ml in absolute ethanol
    Vortex well before using
  4. 1x lysis buffer
    5x reporter lysis buffer     2 ml
    ddH2O                          7.9 ml
    100x protease inhibitor    100 μl

Acknowledgments

This protocol was modified from a crypt isolation procedure reported by Kulkarni and Yielding (1985). This work was supported by RO1 CA10922 from the National Cancer Institute and P20s RR15563 and RR016475.

References

  1. Kulkarni, M. S. and Yielding, K. L. (1985). DNA damage and repair in epithelial (mucous) cells and crypt cells from isolated colon. Chem Biol Interact 52(3): 311-318.
  2. Zeineldin, M., Cunningham, J., McGuinness, W., Alltizer, P., Cowley, B., Blanchat, B., Xu, W., Pinson, D. and Neufeld, K. L. (2012). A knock-in mouse model reveals roles for nuclear Apc in cell proliferation, Wnt signal inhibition and tumor suppression. Oncogene 31(19): 2423-2437.
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Zeineldin, M. and Neufeld, K. (2012). Isolation of Epithelial Cells from Mouse Gastrointestinal Tract for Western Blot or RNA Analysis. Bio-protocol 2(22): e292. DOI: 10.21769/BioProtoc.292.
Q&A

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Ipsita Chatterjee
bar ilan university
Dear Sir,
1. I found a contradiction in the paper that it is mentioned to take 680 mM of Na2hpo4 9.65g for 1 L solution. I will be highly obiliged if you could please make me understand if the final conc. (680mM) is correct or the weigh? (because it should be 96.5 g for 1L if the final con is 680 mM).
2. I tried this protocol once with final concentration of 680mM, but the solution B is crystallizing on ice, since you have mentioned to do it on ice, i would like to ask you, if you also observe the same problem.
3. And it would be really helpful for me to know approximately what is the concentration of RNA you get by this protocol.
11/20/2018 1:36:07 AM Reply
Maged Zeineldin
Molecular Biosciences Department, University of Kansas, USA

Thanks for the questions!
The weight is the correct value (9.65 g/L) and you should not see any precipitate with this concentration.
For the RNA yeild, you should be able to get at least 20-ug RNA by following this protocol.

11/23/2018 6:18:43 AM


Ipsita Chatterjee
bar ilan university

Hi, Sorry for bothering you one more time, I am isolating the RNA with QIAGEN kit, only from large intestine, Last time i barely got any RNA. Could it be beacuse i used a higher concentration of NA2Hpo4? I m guessing you are also isolating RNA seperately from small and large intestine , and for each you are getting minimun 20 ug yield ? Please if you could suggest anything

11/23/2018 6:26:09 AM


Maged Zeineldin
Molecular Biosciences Department, University of Kansas, USA

I assume this was because using the higher concentration. I believe when using the correct concentration of Na Phosphate you will get good amount of RNA. Small intestinal pieces yield more RNA than the colon but I used to get al least 20-ug from the colon as well. However, I have not used Qiagen for RNA extraction in this protocol. I used to use Trizol https://www.thermofisher.com/order/catalog/product/15596026
or TriZol followed by DirectZol for RNA extraction
https://www.zymoresearch.com/direct-zol-isolation-kits

11/23/2018 6:35:36 AM


Ipsita Chatterjee
bar ilan university

Hi, sorry to disturb you one more time, at the end of the procedure there is the centrifuge, in the paper you mention both 1000 g and 1000 rpm for 10 minutes, may be if could please tell me its 1000 rcf (g) or 1000 rpm for 10 minutes?

12/17/2018 2:54:04 AM


Ipsita Chatterjee
bar ilan university

and if you could let me know the RIN of this protocol

12/17/2018 2:54:51 AM


Maged Zeineldin
Molecular Biosciences Department, University of Kansas, USA

Thanks for the questions!
You can use either (Xg) or rpm.
I have not run the RNA on a bio-analyzer to give you the RIN. However, I ran it on agarose gel and it looked good to me.
Good luck!

12/17/2018 5:37:07 AM


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