Preparation and storage method of chicken erythrocyte

A chicken red blood cell and storage method technology, applied in the field of reagents for medical testing, can solve the problems of inability to remove red blood cells, white blood cells, adhered red blood cells and cell fragments, impurity of chicken red blood cells, non-compliance, etc., so as to reduce the cost of cell preparation and avoid complicated Steps, the effect of reducing the CV value

Inactive Publication Date: 2014-01-29
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AI-Extracted Technical Summary

Problems solved by technology

However, this method is relatively rough, and cannot remove white blood cells, adhered red blood cells, and cell debris from the...
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The invention discloses a preparation and storage method of chicken erythrocyte. The method comprises the following steps: collecting heparin anticoagulation venous blood of an adult healthy rooster; purifying by adopting the following steps: removing leukocyte by employing a gelatin sedimentation method; removing synechia among cells by ethylenediamine tetraacetic acid treatment; removing soterocyte and cell debris by employing a low-speed centrifugal process; fixing and storing the purified chicken erythrocyte in a glutaraldehyde solution; dyeing regularly and detecting an X-axis half-peak variation coefficient (CV) of chicken erythrocyte deoxyribonucleic acid (DNA) in flow cytometry detection, wherein chicken erythrocyte deoxyribonucleic acid can be used when X-axis half-peak variation coefficient is within 10%. The chicken erythrocyte CV value obtained by the invention accords with the commercially available standard, is simple and convenient to operate, and strong in repeatability, and can be developed as the commercially available product.

Application Domain

Dead animal preservationBlood/immune system cells

Technology Topic

CELL DEBRISChemistry +11


  • Preparation and storage method of chicken erythrocyte
  • Preparation and storage method of chicken erythrocyte
  • Preparation and storage method of chicken erythrocyte


  • Experimental program(1)

Example Embodiment

[0030] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.
[0031] Unless otherwise specified, the reagents used in the following examples are all analytically pure reagents.
[0032] 1. Extraction and purification of chicken red blood cells
[0033] (1) Store the freshly collected heparin sodium anticoagulated chicken red blood cells temporarily at 4°C and separate them as soon as possible.
[0034] (2) Mix the anticoagulated venous blood with 3% gelatin solution in a ratio of 1:1, and let the solution stand for layers.
[0035] Preferably, the gelatin solution formula is: 30 g of high-quality gelatin, 0.27 g of potassium dihydrogen phosphate, 1.42 g of disodium hydrogen phosphate, 8 g of sodium chloride and 0.2 g of potassium chloride, dissolved in 1 L of deionized water, heated in a boiling water bath Dissolve and store at 4°C until use.
[0036] (3) Remove the upper layer solution, and resuspend the lower layer of red blood cells with diluent. After standing for 10 minutes, centrifuge the cells at 200 rpm for 3 minutes, and remove the supernatant solution. Finally, diluent was added to the deposited cells to resuspend, and 0.3% placental blue exclusion method was used to detect the viability of suspended cells (Dai Jianjun, Rui Rong, Wu Caihong, Ge Lijun, Lu Qingzhu, GV stage oocyte vitrification cryopreservation technology. Journal of Northwest Agriculture and Forestry University (Natural Science Edition), 2007. 35(8): p.34-38), it can be seen that it is basically above 95%. see figure 1 Shown, the morphology of freshly isolated chicken erythrocytes stained with placental blue at a magnification of 400 times.
[0037] Preferably, the dilution formula is: potassium chloride 0.4 g, potassium dihydrogen phosphate 0.06 g, sodium bicarbonate 0.35 g, sodium chloride 8 g, disodium hydrogen phosphate 0.048 g, glucose 1 g and ethylenediaminetetraacetic acid 0.29 g g, dissolve in 1L deionized water, and adjust the pH to 7.2-7.4 with NaOH.
[0038] Second, the fixed storage of chicken red blood cells
[0039] (4) After separation of chicken red blood cells, centrifuge at 1000 rpm, remove the supernatant, add 2.5% glutaraldehyde, and adjust its density to 1×10 7 cells/mL and stored in a 4°C refrigerator.
[0040] Preferably, the formula of the glutaraldehyde fixing solution is: 100 mL of 25% glutaraldehyde, 0.27 g of potassium dihydrogen phosphate, 1.42 g of disodium hydrogen phosphate, 8 g of sodium chloride and 0.2 g of potassium chloride, 900 mL of deionized water Dissolve and adjust the pH to 7.2-7.4 with NaOH.
[0041] 3. Flow cytometer detection and calibration
[0042] (5) Every 30 days, take 100 μL of fresh or fixed chicken red blood cells, add diluent to wash, centrifuge at 200 rpm, and discard the supernatant. Add 1 mL of propidium iodide staining solution to the deposited cells, stain at 37 °C for 20 min, wash with the dilution solution and adjust the cell density to 1 × 10 6 cells/mL
[0043] Preferably, the propidium iodide staining solution formula is: propidium iodide 0.05 g, triton 1 mL, RNase A enzyme 0.05 g, potassium dihydrogen phosphate 0.2 g, disodium hydrogen phosphate 1.42 g, sodium chloride 8 g , 0.4 g of potassium chloride and 0.29 g of ethylenediaminetetraacetic acid were dissolved in 1 L of deionized water, and the pH was adjusted to 7.2-7.4 by NaOH.
[0044] (6) The stained chicken erythrocytes were filtered through a 400-mesh filter and detected by flow cytometer. Taking FL2+H as the abscissa and counts as the ordinate, the CV value of chicken erythrocytes was about 5-7%. , which is lower than the 10% of the commercial requirements, and also better than the 12% CV value reported in the domestic literature. see figure 2. and 3, the CV values ​​of chicken erythrocytes freshly isolated and stored for 90 days were 6.2% and 5.8%, respectively, which proved that they could be used for flow cytometry calibration.


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