Staining system preparation.

, the extraterroral blood staining system preparation 1. Experimental principle Of human exosome blood lymphocytes
culture
method was proposed by Moorhead in 1960. Under normal circumstances, the extraterritorial blood lymphocytes are in the G1 stage (or G0 phase), but in-body to give certain conditions, culture, after 72 h can obtain a large number of silky dividing cells. This simple-to-take, low-blood culture method has been widely used.
plant hemocoagulant (PHA) to the culture, and lymphocytes are stimulated to convert to lymphocytes and enter silky division. After short-term culture, by the autumn daffodil treatment, can inhibit the formation of spindle silk when cells divide, so that cell division stops in the medium term, at the same time it can change the viscosity of the cytoplasm, causing chromosomes to disperse in the cytoplasm. After low seepage and fixation, a large number of chromosomal splitting images with good dispersion effect can be obtained. The body's 1 ml of exomes generally contains about (1 to 3)×106 small lymphocytes, enough chromosomal specimen preparation and analysis. This section introduces the suspended culture of extraterrestorial blood lymphocytes, as well as the preparation of chromosomal specimens.
2.
. Reagents
equipment
(1) 2 ml syringe, culture bottle, scale straw, 15 pounds sterilization 20min. Centrifugal tubes, straws, tubes, slides, washing fluid in accordance with the general cleaning, drying spare.
(2) ultra-clean workbench,
rout thermostat
culture box,
balance
, centrifuges,
microscopes,
and so on.
(3) Reagents:
(1)
Bedle
Preparation:
RPMI 1640 Culture Base 4ml
Maverick
Serum
1ml
Penicillin and streptomycin each 100IU/ml
PHA0.2 ml
Heparin 1 drop
with 5% NaHCO3 to 7.2 to 7.4, 4 degrees C standby.
(2) Heparin: 0.2g is said to be dissolved in 100 ml of double steamed water at a concentration of 0.2% and sterilized.
(3) Autumn daffodils: physiological saline water is made into a concentration of 20 μg/ml, sterilized, divided, set at -20 degrees C.
(4) low seepage: 0.075mol/L KCl.
(5) fixed liquid: methanol: ice acetic acid (3:1), temporary preparation.
(6) Giemsa working fluid: 1 raw liquid and 9 phosphoric acid buffer, temporary preparation.
(7) phosphate buffer: 1/15mol/L Na2HPO4, 1/15mol/L KH2PO4, etc.
(8) 5% NaHCO3 sterilization filter pumping standby.
3. The experimental steps
(1) inoculation and culture.
(1) Under sterile conditions, 0.2% heparin solution (physiological saline preparation, sterilization) is absorbed by sterilizing syringe 0.2 ml, after wetting the syringe, the intravenous blood is taken 1 to 2 ml, the syringe is turned so that the blood and heparin are fully mixed.
(2) Under sterile conditions, heparinized blood is dripped into the outer blood culture, and 28 to 30 drops (needle No. 7) are dripped into each 5 ml of culture fluid and gently mixed.
(3) in a 37-degree-C thermostat, static culture 68 to 72h. Note that the next day to observe the culture of blood clotting, hemolysis or long bacteria phenomenon, can shake the culture fluid every day so that the cells are fully cultured.
(4) 2 to 3h before the end of culture, add the concentration of 20 μg/ml of akiletin, each 5 ml medium with 7 needles, plus 3 to 4 drops to make the final concentration of 0.1 μg/ml. Gently shake well, then put into the thermostat box, continue to cultivate 2 to 3h, in order to accumulate more stop in the medium-term split elephant.
(2) production.
(1) Harvest cells: take out the culture bottle from the thermostat, blow the bottle wall with a straw, so that the cells are all out of the bottle wall, and then move the cell fluid into the conical centrifuge tube, 1500 l/min, centrifugal 10min, to dehydrate.
(2) low seepage treatment: add 0.075mol/L KCl 8ml at a pre-temperature of 37 degrees C, after repeated blowing (about 100 times), set 37 degrees C water bath in the low seepage treatment of about 25min.
(3) pre-fixed: add fresh preparation fixation liquid 1 ml (methanol: ice acetic acid: 3:1), gently mix.
(4) centrifugation: 2000 l/min, centrifugation 10min, absorption of liquid.
(5) fixation: slowly add 8 ml of retaining fluid along the centrifugal tube wall, gently mix well, fix 10min.
(6) centrifugation: i.e., suck up the liquid.
(7) and then fixed: add 8 ml of fixation liquid, mix well, fix 10min.
(8) centrifugation: i.e., suck up the liquid.
(9) cell suspension: according to the amount of cells, add the appropriate amount of fixed liquid, fully mixed, made into cell suspension.
(10) drops: take out the pre-soaked ice water slide, use a straw to absorb mixed cell suspension at a distance of about 30 cm away from the ice tablets for drips, each drop of about 2 to 3 drops of cell suspension, so that the cells are better dispersed. Generally, each culture bottle can produce 3 to 5 specimen pieces.
{11} Dyeing: After the specimen is dried, it can be dyed with dye (1 part of Giemsa liquid, 9 parts of pH6.8 phosphate buffer) and 15min after flushing of tap water (flushing from the back).
(3) Mirror examination: each human body cell has 46 chromosomes, depending on the length of the chromosome and the location of the silk particles, can be divided into 22 pairs of normal chromosomes and a pair of sex chromosomes, men are 46, XY, women are 46, XX. chromosomal specimens prepared for in-body cultured cells are prepared 1. In-body cultured cells, when more dividing cells (24h, round shiny cells) were observed under the inverted mirror, an autumn daffodil solution was added with a final concentration of 0.3 μg/ml culture;
2. Continue to train 3h;
3. Pancrease digests and collects cells into centrifugal tubes;
4. Centrifugal 1000 l/min, centrifugal 10min, go to
5. Hanks liquid wash, centrifuge, go to clear;
6. Low seepage treatment: add 0.075mol/L KCl solution 8 ml, put 30min in a heated water bath at 37C, blow slightly with a straw (i.e.);
7. Fixation and preparation of chromosomes such as exosomes. 3, Precautions 1. Autumn daffodil treatment time is too long, many dividing cells, chromosomes are short, on the other hand, less and slender, so the concentration and time of autumn daffodils should be accurately grasped.
2. The fixing fluid should be made temporarily before use.
3. The slides must be clean, otherwise the chromosomes are not dispersed well.
4. Chromosomal division index is low: the patient is in the non-active period (release, chemotherapy period), the media nutrient content is poor, the media pH is low or high, PHA is excessive or insufficient, the calf serum quality is not high, the culture box temperature is low, the autumn daffodil treatment time is too short.
5. The fresher the blood sample, the better.
6. During the culture process, if blood-like coagulation is found, the culture bottle can be gently oscillated so that the clots disperse and continue to be cultured back in a thermostat at 37 degrees C.
attached:
. Carnoy fixation fluid: An important feature of
fixation fluid is the ability to penetrate cells quickly, secure them and maintain the integrity of chromosomal structures, as well as the ability to enhance the alkalinity of chromosomes for excellent staining results. Simple fixation fluids are generally difficult to meet these requirements, so two mixed fixation fluids are used in experiments. Carnoy is known as Carno's fixation fluid because of the methanol and ice acetic acid mixture it first used.
Carnoy fixed liquid (methanol: ice acetic acid : 3:1) each time before use need to be temporarily made, long-term placement affects the fixed effect, fixed time 15min to 24h, refrigerator, room temperature can be. If necessary, the ratio of methanol and ice acetic acid can be changed, the proportion of ice acetic acid increased, which is good for cell expansion, chromosomal spread, but easily lead to cell rupture, chromosomal dispersion.
hypotonic solution
low seepage fluid refers to the osmotic pressure and ion strength are lower than normal cell physiological conditions of the solution, permeable pressure is lower than
tissue
solution, mixed with the outer blood, water quickly into the cell, so that it swells, or even ruptures, to obtain a well-dispersed chromosomal division image. Common low seepage: water, low seepage sodium citrate or sodium chloride, potassium glycerate (0.65mol/L), potassium chloride (0.075mol/L), etc.
the low seepage depends on the chemical composition of the low seepage, the temperature of the low seepage and the processing time. Low seepage processing allows cells to expand chromosomes by reverse osmosis, while dispersing nuclear kernels attached to chromosomes so that all chromosomal morphology can be observed on a single plane.
0.075mol/L KCl is generally used in laboratories for low seepage, the advantages of which are: (1) chromosome contouring is clear, can be dystyned, dyeing time is short. (2) For ribbon dyeing can fully display the characteristics of the band type. The low seepage treatment is 37 degrees C, 25 to 30min, whichever is the pre-experimental condition.
giemsa dye
. Giemsa original liquid preparation: called Giemsa powder 0.5g, add a few drops of glycerine grinding, and then add glycerine (so that the total amount of glycera added is 33ml). Keep warm from 90 to 120min at 56 degrees C. Add 33ml methanol and store in a brown bottle. When used, dilute with PBS as required, generally diluted 10 times.
.