小鼠脾脏细胞培养

PRIMARY CULTURE OF MOUSE SPLEEN CELLS & IDENTIFICATION

OF LIVE OR DEAD CELLS

BY DIFFERENTIAL STAINING

Qianqian Chen(118627140313)

Team members: Qingqing Yu,Qing Zhang

April 7th 2012

Abstract

Cells from muti-cellular organism can be isolated and cultured alive in vitro,and keep its physiological and biochemical properties.To vertify it ,our team cultured the mouse spleen cells in vitro and fond that though the cells were microbiological contaminated, the cells somehow survived,thus we proved the conclusion.

Considering our cells were microbiological contaminated we count the cells from other teams to learn the metheod of counting cells to keep the experiment completed. Introduction

Cells culture can be divided into primary culture,subculture and the formation of cell lines.Cells dierctly isloted from organism without proliferation in vitro are called primary cultures.If primary cultures continue to divid and proliferate to form more cells,then the cells will be called subcultured ually the subcultred cells will go to death after a finite numbers of generations in vitro,this prcess is called cell senescence,however if the subcultred cells are transfored and become immortal, then they will become cell lines. According to the difference of cell membrane permiability from the death and the survives,some dyes can permeate the membiane of the live cells(0.15% Eosin Y),but

some can not(1% (w/v) Trypan blue). All cells can be coloured by 0.15% Eosin Y,but only the dead cells can be coloured by 1% (w/v) Trypan blue. Thus 1% (w/v) Trypan blue and 0.15% Eosin Y can be used to distingwish the dead cells from the live cells . Materials and Methods

Methods

1.Sacrifice mouse by neck-breaking (cervical dislocation).

2. Soak the mouse in 70% ethanol for a few minutes.

3. Lay the mouse face up, wet the abdomen with water, lift the out skin with forceps and cut a horizontal opening with scissors, tear the out skin apart to expose the muscle layer. Cut the muscle layer open in a V shape and lift up to expose peritoneal cavity.

4. Push aside the stomach in the upper-left side of the abdomen to expose the sack-shaped, dark-red spleen. Cut off the spleen with scissors and put it into the glass dish.

5. Inject 0.2ml PBS with the L-shape needle into the spleen along the longer axis. Spleen will bloat and become pale. * Not to penetrate the spleen.

6. Punch some holes with the needle on the spleen and scratch it so that spleen cells will flow out to the PBS. At the end, spleen goes from red to yellow and PBS will turn into red. Discard the remaining of the spleen.

7. Blow the remaining PBS with spleen cells with pipette several times to dispense cell clumps into individual cells.

8. Transfer cell suspension to a microcentrifuge tube. Add PBS to 1ml total volume and spin at 500g for 5 min.

9. Discard supernatant. Resuspend cell pellet with 1ml cell culture medium and transfer to the plastic culture dish, which has already 2 ml .

10.Rock the fish to separate cells evenly.

11.Culture in the incubator at 37°C for o ne to two weeks.

12.Observe cultured cells by naked eyes and under the Inverted Microscope .

13.Tap the dish to release attached cells and swirl to the dish to resuspend all cells .

14.Take 0.5 ml of cell suspension and add one drop of 1% Trypan blue or 1.5ml 0.15%

Eosin Y. Mix gently, and stain at room temperature for 2 minutes.

15.Put coverslip over the counting chamber.

16.Pipette a drop of the cell suspension on the V slot on the hemocytometer. The

suspension will flow to the couting area by capillary action.

17.Under the microscope, count live and dead cells in 4 corner and 1 center medium

squares.