Mouse Tissue
Collection
Contact: Ometa Herman
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Materials |
Qty |
Location |
Order Info |
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15mL conical tubes |
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B-011 |
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Staining media (mRPMI (pH 7.4) + NCS) |
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scissors, forceps |
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In Animal room |
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10cc syringe, 25 5/8 needle |
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B-011 |
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Nylon filters |
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Nicole’s Bench B-011 |
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Frosted slides |
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Nicole’s Bench B-011 |
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ACK lysing buffer |
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Fridge # 5 B-011 |
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NCS |
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Freezer # 3 B-010 |
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Collecting
Peritoneal Cavity Cells
1)
The staining
media you use should not have EDTA or azide in it. NCS only.
2)
Prior to
working with the animals, fill a 10cc syringe (equipped with a 25 5/8 guage
needle) with 7cc media and 1cc air for each mouse. Also have prepared a Pasteur
pipet and bulb prepared.
3)
Sacrifice the
mouse using CO2
4)
Using flat
scissors, snip the epithelium of the mouse’s stomach.
5)
Use horizontal
force to pull the skin away from the peritoneal wall.
6)
Using tweezers,
lift up the wall and inject the media and air
7)
Shake the mouse
loosely.
8)
Use the Pasteur
pipet to puncture the peritoneal wall and withdraw
9)
Of the 7mL in,
you can usually obtain at least 5mL out
10) Filter over nylon sheet and count.
Collecting Spleen
Cells
1)
Before
collecting organs, prepare 2 frosted slides by grinding away loose glass debris
in media. Wash with media to clear loosened debris.
2)
Collect spleens
from the mice. Place in a 15mL tube with about 7mL media.
3)
Back in the
lab, empty 15mL tubes into a small petri dish.
4)
Use the frosted
slides to loosely grind the spleen cells out of the spleen into the media.
5)
Filter the cell
suspension over a nylon filter.
6)
Spin 8000rpm
for 3 min
7)
Aspirate.
8)
Resuspend cells
in 2mL ACK lysing buffer. Let stand for 1minute.
9)
After 1 minute,
dilute by adding 10mL staining media.
10) Underlay the suspension with a 9-inch Pasteur
pipet filled with NCS.
11) Spin 8000rpm for 3 min
12) Resuspend cells in 1mL and count in the
hemacytometer using a 1:20 dilution.