Lymphocyte Culture Medium

Product Name

Lymphocyte culture medium

Packing Specifications

5mL/ bottle, 24 bottles / box

Intended Use

It is only used in cell proliferation culture without the function of cell selection, induction and differentiation.The cultured cells were only used for in vitro diagnosis.

The principle

Lymphocyte medium is specially developed for the proliferation culture of lymphocytes in peripheral blood.If lymphocytes need to be cultured in vitro, necessary growth conditions should be added to the outside world, and the lymphocyte culture medium produced by our company can meet the conditions for the in vitro growth.

Main components

Storage conditions and term of validity

-20 ºC preservation, valid for 2 years.

Applicable instrument

Carbon dioxide incubator.

Sample requirement

Peripheral blood about 2 ml.

Method of calibration 

1. Experimental materials

Lymphocyte medium(specification: 5mL/ bottle)

Wright’s stain solution: put 100mg Wright’s stain in the mortar, add a small amount of methanol to grind to no granules, and prepare 0.1% dye solution with methanol.

40 mg /mL colchicine

Low seepage: 0.075mol/L of KCl.

Fixed solution: methanol: glacial acetic acid =3:1.

JiM, mother liquor: say 0.5 g JiM powder in a mortar, measuring 33 ml glycerin, take a small amount added to mortar and JiM powder grinding to no particles together, and then pour the rest of the glycerine, 37 ºC water bath after mixing heat preservation after 2 h, then add 33 ml methanol, stir well after stored in the brown bottle. Set aside.

0.1mol/LpH7.4 phosphate buffer.

2. Operation methods and procedures

2.1 cell pretreatment was observed at the conversion rate

Steps: take lymphocyte culture bottle bottle, under aseptic conditions with 1 ml syringe add 20 blood cells respectively, gently shake well, add 37 ºC carbon dioxide 72 h is placed in the incubator.The cell culture solution was put into 10ml centrifuge tube with a pipette, centrifuged (1000rpm, 5min), the supernatant was discarded, a drop of cell suspension was put on the glass slide, and the slide was pushed.When dry, the cells were stained with ruishi stain, and the cells were sky blue.200 monocytes (both transformed and untransformed cells) were counted under oil microscope.

Judgment of transformed and untransformed cells:

Untransformed cells: small in size, 6-8 cytoplasm in diameter, non-enlarged nucleus, dense chromatin, no nucleolus, and minimal cytoplasm.

The transformed cells are large at 12-25 m in diameter, with enlarged nuclei and abundant cytoplasm.

The conversion rate is calculated as follows:

Cell conversion rate (%) = number of transformed cells x 100%

Total number of cells

2.2 chromosome observation precellular processing

2.2.1 take 2 bottles of cell culture media, under aseptic conditions of 25 blood cells with 2 ml syringe drops, gently shake well, add 37 ºC carbon dioxide incubator in 68-69 – h.

2.2.2 colchicine treatment: after 68-69h treatment, colchicine was added to the cell culture medium bottle with 1ml syringe to achieve a final concentration of 0.4mb g/ml and a total time of 72h after placement for 3-4h.

2.2.3 cell isolation and fixation: the cell bottle was removed and cell culture medium was transferred into 10ml centrifuge tube with a pipette for centrifugation (1500rpm, 7min).Pipetting gun put on clear liquid, add 8 ml of 0.075 mol/L KCl low drainage, gently suck, 37 ºC water bath 25 min.Then add 2ml fixative solution, mix, pre-fixation for 15min, and then centrifuge (1500rpm, 7min).The supernatant was removed with a pipette, and 8ml of the fixative solution was added. The supernatant was gently absorbed and set for 15min before centrifugation (1500rpm, 7min).Carefully discard the supernatant, add 8ml of the fixative solution, gently absorb well, and let it rest for 15 minutes or overnight.

2.2.4 preparation of suspension: centrifuge (1500rpm, 7min), carefully absorb and discard the supernatant with a dropper, and leave a little supernatant as the cell remains.

2.2.5 drip plate: the cells were mixed with a straw, and the cell suspension was dropped from 10 to 20cm high on a clean glass slide, which was cooled by ice in advance, and gently blown away. The cells were dried slowly over the flame of the alcohol lamp or dried naturally at room temperature.

2.2.6 dyeing: 0.1mol/L pH7.4 phosphate buffer was diluted with 1:10 to filter the dye solution.Add a few drops of dye onto the glass slides, stain for 5-10min, rinse with 0.1mol/L pH7.4 phosphate buffer, remove the excess dye, and dry.Observe under oil mirror.Results: the cell morphology was good, the cell nucleus was large, the chromosomes were dispersed and clear.

Positive judgment value or reference range

The cell morphology is good, the division cell is many, the cell karyotype is big,the chromosome is

clear.

Interpretation of test results

Because of the differences between individuals, lymphocyte growth rate has a slow, users need to adjust the culture time.

Limitations of testing methods

This product requirement for cell culture condition of 37ºC, 5% CO2, cultivate saturated humidity conditions.

Product performance index

pH                        7.0±0.5

Osmotic pressure             280-340 mOsmol/kg

Endotoxin                  ≤0.5 EU/mL

Sterile                   Clarification of medium after 14 days of culture by membrane filtration

Appearance                  Reddish clear water solution

The load                    5mL±0.3mL

Matters needing attention

1. This product is only used for in vitro diagnosis, not for treatment.

2. Use of expired products is strictly prohibited.

3. – 20 ºC, valid for 2 years.Avoid microbial contamination.

4. Remove the culture media from the refrigerator, restore to room temperature and shake well before use.

5. The product is free of harmful substances and can be cleaned by contact with skin water. If it enters human eyes, it should be rinsed with plenty of water.

6. The product will be treated as clinical waste after use.

7. This product requirement for cell culture condition of 37ºC and 5% CO2, saturated humidity conditions, otherwise it will affect the quality.