Ready-to-use medium, includes all the necessary substances for cultivation of whole blood lymphocytes. It is intended for cultivation of blood lymphocytes in vitro in order to obtain dividing cells and subsequent analysis of metaphase chromosomes.
"lymphokar-2" medium is auxiliary tool in the diagnosis of possible pathological disorders at the genetic level. It is used for in vitro diagnostics (karyotyping) - for cultivation of blood lymphocytes in vitro and subsequent cytogenetic analysis of metaphase chromosomes with mitotic index calculation.
The analysis is carried out using micromethod of cultivation of human peripheral blood lymphocytes and preparation of chromosomes metophase. Heparinized blood is used for analysis.
Ready-to-use serum-free medium, includes all necessary substances for cultivation of whole blood lymphocytes: a mixture of inorganic salts, amino acids, vitamins, glucose, phenolic red, antibiotics, as well as PHA-P, etc.
The presence of phytohemagglutinin-P (PHA-P) in the medium at the optimal concentration for human lymphocytes provides mitogenic effect of the medium.
Stopping mitotic division with subsequent accumulation of metaphase plates is achieved by incubation of cells with colchicine. In the future, for cytogenetic analysis, the cells are transferred to hypotonic solution of potassium chloride, fixed with a mixture of methanol with glacial acetic acid.
METHOD OF APPLICATION:
Before direct application, the medium should be thawed. Defrosting should occur naturally in the refrigerator at temperature of +2 to +8 ° C. Accelerated defrosting of the medium and thawing at temperature of 37 ° C is prohibited. It is recommended to avoid multiple freeze-thaw cycles.
After defrosting and stirring, the medium is clear liquid of red-pink color.
Getting to work, mix the contents of each bottle carefully by gently turning the bottle over.
Opening the vial and perform subsequent manipulations under aseptic conditions.
For cultivation of 0.5 ml of blood, it is recommended to use 7 ml of medium.
In a sterile container (vial, test tube, etc.) with capacity of at least 10 ml, add 7 ml of medium under aseptic conditions.
Lymphokar-2 cat. no.H11 and no.H212 with volume of 60 and 100 ml must be transferred to sterile containers with volume of at least 10 ml.
Lymphokar-2 cat. no.H10 is available in ready-to-use volume of 7 ml test bottle. The cultivation of lymphocytes is carried out directly in the test bottle without transfer to other containers.
Add 0.5 ml of whole heparinized blood to the container with the medium.
Incubate sterile containers with test samples in a thermostat at 37 ° C for 72 hours.
Dissolve colchicine (0.1 mg/ml) in 5 ml of distilled water. 1.5 - 2 hours before the end of incubation, stop cell division by adding 50 ml of colchicine aqueous solution to each container with cell culture (test bottle, test tube).
1.5-2 hours after colchicine is added, shake the container and transfer its contents to a centrifuge tube. Cells should be precipitated by centrifugation for 5-7 minutes at 1000-1500 rpm.
Prepare hypotonic potassium chloride solution (0.075 M) by dissolving 0.56 g of KCl in 100 ml of distilled water. Preheat the solution to 37 ° C.
After centrifugation, remove supernatant, break up precipitate and add 10 ml of heated hypotonic KCl solution to the test tube. Mix the precipitate well and place the test tube in a thermostat, incubate at 37° C for 10-12 minutes.
The treated cells should be precipitated by centrifugation for 5-7 minutes at 1000-1500 rpm.
The supernatant should be selected and removed, leaving 0.5-1.0 ml of the infusion fluid, and resuspend the cells with vigorous shaking.
Next, the cells are fixed with a freshly prepared mixture of methanol and acetic acid in a ratio of 3:1. In a test tube containing suspension of cells, add fixing solution (2-3 ml) with a sharp jet, and bring the volume in the test tube to 6-8 ml. Break up the sediment and put the test tube in the refrigerator for 5-10 minutes.
Cells treated with fixing solution must be centrifuged at 1000-1500 rpm for 5-7 minutes. Remove the supernatant, add 8-10 ml of cooled fixing solution to the sediment. Break up the sediment. Then stand at 4 ° C for 15-20 minutes, placing in the refrigerator.
Cells treated with fixing solution should be centrifuged at 1000-1500 rpm for 5-7 minutes. Remove the supernatant. Repeat the fixation and cells treatment with a freshly prepared mixture of methanol and acetic acid in a ratio of 3:1 (add fixing solution (2-3 ml) to a test tube with suspension of cells, and bring the volume in the test tube to 6-8 ml). Break up the sediment and put the test tube in the refrigerator before staining the tested samples.
The treated cells should be precipitated by centrifugation for 5-7 minutes at 1000-1500 rpm. Remove the supernatant. Resuspend the precipitate in 0.5-1.0 ml of fixing solution and dig out the suspension on cooled, water-soaked slides. Then, in order to fix the glass, quickly hold it over the flame of the burner or dry it at room temperature.
In conclusion, the preparations are subjected to artificial "aging" and stained using Giemsa dye (or another dye with similar characteristics) according to the generally accepted method.
This Instruction is developed on the basis of recommendations for development of chromosomal preparations (Methodical manual for doctors "Cytogenetic methods of chromosomal diseases diagnoses", Moscow, 2009).
HOW SUPPLIED: in hermetically sealed plastic bottles (PET containers) of 7, 60 or 100 ml. Euroflakons with a wide neck.
Shelf life: 2 years.
The product shelf life after the initial bottle opening is 1 month from the date of opening at temperature of +2 to +8 ° C.
Store in a place protected from light at temperature of -10 to -20 ° C.