Nov 10, 2022
Establishment of primary intestinal epithelial cells and leukocytes from the three-spined stickleback, Gasterosteus aculeatus
- abdelmounaim nouri1,
- Maria L. Rodgers2,
- Daniel L. Bolnick2,
- Natalie C. Steinel1
- 1Department of Biological Sciences, Olsen Hall 224, University of Massachusetts Lowell, MA, USA.;
- 2Department of Ecology and Evolutionary Biology, University of Connecticut, CT, USA.
Protocol Citation: abdelmounaim nouri, Maria L. Rodgers, Daniel L. Bolnick, Natalie C. Steinel 2022. Establishment of primary intestinal epithelial cells and leukocytes from the three-spined stickleback, Gasterosteus aculeatus. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqj565vk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 26, 2022
Last Modified: November 10, 2022
Protocol Integer ID: 70490
Keywords: Three-spined stickleback, Primary cell culture, Epithelial cells, Leukocytes, Enzymatic digestion
Abstract
This protocol details the Establishment of primary intestinal epithelial cells and leukocytes from the three-spined stickleback, Gasterosteus aculeatus.
Attachments
535-1117.docx
30KB
Materials
Stock solutions:
0.1M dithiothreitol:
| A | B | |
| DTT powder | 155 mg | |
| dH2O DNase free | 10 mL | |
| Aliquot in 1ml tubes | ||
| Store at -20 C | ||
Note
Highly unstable at RT.
Mucus removal solution:
| A | B | |
| 1X HBSS | 18.6 ml | |
| DTT solution | 1 ml | |
| FBS | 0.4 ml | |
| Divide into two 15 ml tubes | ||
Epithelial cells recovery solution:
| A | B | |
| EDTA | 29.224 g | |
| FBS | 0.4 ml | |
| HBSS | ||
| Adjust pH to 7.3 using hydrochloric acid or sodium hydroxide. | ||
Enzymatic digestion solution:
| A | B | |
| LiberaseTM | 1.4 Wünsch units/ml | |
| DNase I | 24 U/ml | |
| 1X HBSS | 7 ml |
Reagents:
Dithiothreitol (DTT)Thermo Fisher ScientificCatalog #R0861
Cell Dissociation Buffer enzyme-free Hanks Balanced Salt SolutionThermo Fisher ScientificCatalog #13150016
Ethylenediaminetetraacetic acid 99% pureThermo Fisher ScientificCatalog #AC118432500
Thermo Scientific™ Deoxyribonuclease I bovine pancreasThermo Fisher ScientificCatalog #AAJ62229MB
Supply Solutions Liberase™ DL Research Grade low Dispase concentration 2x5mgFisher ScientificCatalog #501003356
Leibovitz's L-15 MediumThermo FisherCatalog #11415064
Corning™ Penicillin-Streptomycin SolutionFisher ScientificCatalog #MT30002CI
Corning™ Premium Fetal Bovine SerumFisher ScientificCatalog #MT35015CV
Leuko Spin MediumpluriSelectCatalog #SKU 60-00091-10
Equipment:
Equipment
Centrifuge
NAME
Eppendorf
BRAND
5804
SKU
Equipment
Fisherbrand™ accumet™ AE150 Benchtop pH Meter
NAME
pH meter
TYPE
Fisherbrand™
BRAND
AE150
SKU
LINK
Equipment
Cole-Parmer® INC-250 Series Mini CO2 Digital Incubator
NAME
Digital Incubator
TYPE
Cole Parmer
BRAND
71717
SKU
LINK
Equipment
Microscope: Leica DMi1
NAME
Microscope
TYPE
Leica
BRAND
DMi1
SKU
LINK
Stereoscope: Leica S7E
Equipment
Stereoscope
NAME
Stereoscope
TYPE
Leica
BRAND
S7E
SKU
LINK
- Biosafety cabinet: Sterilgard III Advance
Before start
Fish dissection:
- Prepare one Petri dish On ice with 10 mL of 1X PBS for collecting the tissue.
- Prepare Two Petri dishes containing 10 mL of PBS 1X, 0.1% povidone-iodine and, two Petri dishes with 10 mL 1X PBS and place inside the biosafety cabinet to sterilize the intestine.
Fish dissection
Fish dissection
After following approved euthanasia procedures, place the fish’s body On ice .
Make a ventral incision from the cloaca to the jaw using sharp surgical scissors.
Make two lateral incisions just behind the opercular flaps down to the lateral line of the fish.
Using two pins, secure the fish on its dorsal side on a dissecting pad.
To detach the intestine, make a cut at pyloric caeca on one side and the cloaca on the other side.
Place intestine into a Petri dish containing 10 mL of cold 1X PBS.
Using forceps and mini dissecting scissors, open the intestine by making a longitudinal incision.
Bring the Petri dish containing the intestine into the biological safety cabinet and wash the intestine by submerging in two successive 0.1% povidone-iodine washes for 00:05:00 each.
5m
Wash twice for 00:05:00 each in a Petri dish containing 10 mL of cold 1x PBS to remove iodine.
5m
Mucus removal:
Mucus removal:
Transfer opened gut in 10 mL of mucus removal solution and incubate for 00:10:00 at 17 °C on a gyratory rocker for 00:10:00 .
20m
Resuspend the gut tissue in a fresh 10 mL of mucus removal solution and incubate again incubate for 00:10:00 at 17 °C on a gyratory rocker.
10m
Epithelial cells recovery and enzymatic digestion:
Epithelial cells recovery and enzymatic digestion:
Note
Enzymatic digestion is affected by the temperatures.
Transfer the gut tissue to 10 mL of epithelial cells recovery solution and incubate for 00:10:00 at 17 °C on a gyratory rocker.
10m
In order to recover epithelial cells in the suspension, remove the gut tissue from Epithelial cells recovery solution and keep it On ice for enzymatic digestion step. Centrifuge the cell suspension at 300 x g for 00:10:00 at 17 °C .
10m
Remove the supernatant and resuspend the epithelial cell pellet in HBSS with 2% FBS and 1% Pen Strep.
Transfer the intestinal tissue from step 13 to 7 mL of enzymatic digestion solution, then incubate for 00:30:00 at 17 °C on a gyratory shaker.
30m
Collect and save the cell suspension at 17 °C .
Resuspend the remaining intestinal tissue removed from the enzymatic digestion solution in 7 mL of fresh enzymatic digestion solution for a second enzymatic digestion.
Incubate for an additional 00:30:00 at 17 °C on a gyratory shaker.
30m
Recover the cell suspension and pool with cell suspensions obtained from step 16 in a 15 ml conical tube and keep at 17 °C .
Filter the obtained cell suspension through a 40 µm mesh cell strainer into a new tube to remove cell clumps.
Ccntrifuge the obtained unicellular suspension at 300 x g for 00:10:00 at 17 °C .
10m
Resuspend the cell pellet in 5 mL L15 with 2% FBS and 1% pen Strep.
Density gradient:
Density gradient:
Use double-density leukocyte isolation medium to recover all leukocytes from the cell suspension.
In a 15 mL conical tube, add 10 mL of the density medium.
Carefully layer 5 mL of the cell suspension onto the density medium and mix the two phases.
Centrifuge for 00:20:00 at 750 x g at 17 °C .
20m
After the density centrifugation, one white layer of cells appears between the L15 medium and Ficoll. Aspirate the top layer of the L15 medium.
Next, transfer the mononuclear and polymorphonuclear cell layer to a new conical tube, while making sure to not aspirate the Ficoll gradient with the cells. Wash the cells by centrifuging them at 17 °C , 300 x g , once with 10 mL of L15 2% FBS and 1% PenStrep.
Cell seeding:
Cell seeding:
Seed the cells into 96 wells plate at a density of 1x106 cells/ml in L15 media with 10% FBS and 1% PenStrep.