Newborn screening of bovine serum
Experimental purposes
Learn of new bovine serum in cell culture, grasp the role of serum screening techniques.
Experimental principle
Bovine serum is one of the Chinese medicine ingredients cell culture. In a medium stir-in serum (10%), the first can add cell growth needed growth factor, hormone, 1 attached factor etc, then it with detoxification effect, can remove fatty acids, heavy metal and some protease toxicity, Again, it still can suspend pancreatic enzyme function, and protect cells against mechanical damage, etc. But serum complicated composition, different sources, different batches of serum for cell growth production function of difference is bigger. Serum storage life only one year, different storage period of serum effectiveness is different also. So, when the new purchase in the serum or start a major test of time, should first bovine serum screening.
Screening, usually choose normal diploid cells and not too good raised the tumour cells, also can make about to the cultured cells. In addition, it is best known quality serum as control group.
Equipment, materials and reagents
instrument
The CO2 incubator, anti-oil workbench, trace pipette, sterilization pot
materials
96 hole training board, scale straws, pipette, tube, straws, liquid cylinders, blood count plate, HeLa cells, Mel cells
reagent
DMEM, different batches of serum, pancreatic enzyme, double resistance
Experimental steps
1. Take HeLa cells and Mel cells (originally should take the normal diploid cells and not too good raised tumor cells, but the laboratory condition is limited, HeLa 19.88% do cell), trypsin digestion, machine-brushing serum-free media (double antioxidant) beforehand percussion into cell suspension liquid (avoid before serum interference). Counting.
2. Take around 4,000 HeLa cells to join the first row holes (in plus before concussion medium). This experiment by counting after should add 11 muon L cells.
3. In A and H line first hole joining 89 muon L containing standard serum (2004.12) medium. (control group)
4. In A and H line each hole joining 100 muon L containing standard serum levels of medium.
5. Will first of fluid absorption pore 100 muon L to second hole, from 2nd hole suction 100 muon L to third hole, then the same. The last 100 muon L rejected. Formed so from the first to the last one hole hole of cells from about 2056,1028 until gradient 1. (to blow fine uniform)
6. Each hole again add 100 muon L containing standard serum levels of medium. This can guarantee the hole nutrition.
7. In the B do take the same method. Different is with contain need for the screening of the batch number 011217 serum instead of standard serum.
8. In C to G line using the same method. Serum batch number is 030224,040528,040830,041012,041016 respectively.
9. Will 96 hole training board into CO2 incubator 37 ℃ training for a week.
10. Training ended, observe cell growth. Comparison in the same cell concentration of students for cell growth not clear the influence. In the same cell concentration. Under the maximum number of cell growth medium contains serum are then used as selected serum sample.
Experimental results
From the first five elements start counting, result see table below
JiLa number line 5 line 6 line 7 line 8 line 9 line 10 line 11 line 12
A 00 00 00 00 00 00 00 00
B 22 12 03 03 01 00 00 00
C 42 17 04 05 01 01 02 00
D 15 04 04 00 00 00 00 00
E 14 07 00 03 02 00 01 00
F 25 10 09 03 00 00 00 00
G 24 15 00 01 00 00 00 00
H 00 00 00 00 00 00 00 00
Experiments, we can see the group C cells JiLa number is the most (relative to the same column of speaking). Group C cells is to use for 030224 serum batch to develop and this showed no more can promote 030224 serum HeLa cell growth. In the other students did experiments, is group E Mel cells grow best, this explains 040830 more can promote Mel cell growth. From that, the different cells are adapted to different levels, cannot treat as the same. A group and control group H is, but did not grow cells JiLa. The situation in the class exists in the control group, is probably the serum there are problems or while doing an experiment didn't add serum.
Experimental discussion
Bovine serum is cell cultures important to cultivate ingredients, one of the growth of cells have extensive effect. In cell culture, we often join 10% of serum. Serum levels of quality of cell culture is critical to the success of. Generally speaking, bovine serum include the following components: growth factor (such as hormones, interleukin etc), they can promote cell growth, Stick add factors, they can promote cells stick wall, often only cells stick wall to value-added (suspension cultured cells except), Protein (e.g., serum albumin, globulin, etc.), either as nutrients, and may suspend pancreatic enzyme action; Many components of unknown material. Serum still can rise to detoxicification, such as fatty acid, heavy metal and some protease toxicity. Serum also may be the cells from mechanical damage.
Different manufacturer, different batch production of quality is different from the serum cell growth in different effect, so get a new serum or start to significant scientific experiment, want to undertake newborn screening of bovine serum. Some serum may have mycoplasma pollution; Different serum of different cells; the promoting function of different Serum placed after period of time, nutritional value can be reduced, so it is necessary for newborn screening of bovine serum. Newborn bovine serum is most appropriate test cell growth serum can be used in long-term experiments.
This experiment is a plus all the cells. This makes each row of cells to minimize total differences, so as to make the system error decreases, Moreover, this method is convenient, the efficiency has also improved, Experimental results show that our method is relatively superior this.
