Preparation and analysis of reticulocyte samples
Preparation and analysis of reticulocyte samples
This experiment is based on "Color Atlas of Practical Flow Cytometry", edited by Shukui Wang and Zhenying Zhou.
Operation method
Preparation and analysis of reticulocyte samples
Principle
Reticulocytes are incompletely mature red blood cells. In the process of cell maturation, the RNA content in its cytoplasm is gradually replaced by hemoglobin, therefore, the detection of the amount of RNA in the red blood cell represents the maturity of the red blood cell. Thus, it is an important method to detect reticulocytes. The reticulocyte count can determine the hematopoietic status of the bone marrow erythrocyte system. In hemolytic anemia, due to the large number of reticulocytes entering the circulation, reticulocytes can be as high as 0.20 or higher. Reticulocytes peak 5-10 d after acute blood loss and return to normal after 2 weeks. In typical cases of aplastic anemia, the reticulocyte percentage is often 0.005. A reticulocyte count of less than 5 X 109/L is one of the criteria for the diagnosis of aplastic anemia. Elevated reticulocytes indicate active erythropoiesis in bone marrow, which is common in various acute and chronic hemorrhagic hemolysis and various types of proliferative anemia with effective treatment, such as iron-deficiency anemia, hemolytic anemia, megaloblastic anemia, and so on, and it is a very high-value indicator for monitoring in the treatment of anemia. Hypoplasia of reticulocytes is seen in various diseases with low bone marrow proliferative capacity, especially in severe aplastic anemia.
Materials and Instruments
Whole Blood Move 1. 0.5 ml of whole blood is drawn and injected into 0.5 ml of 0.1 mol/L EDTA solution. For more product details, please visit Aladdin Scientific website.
EDTA Good Buffer Sodium Citrate Buffer Glutaraldehyde Buffer PBS Pyronin Y Working Solution
2. 50 uI of EDTA anticoagulated blood is taken with a microfuge, placed in another centrifuge tube, and 5.0 ml of Good buffer is added, centrifuged at 1000 r/min for 5 min, and washed three times consecutively.
3. Precipitated cells are added to 1.0 ml of sodium citrate buffer, and suspended by gentle shaking. 4. The suspension is slowly injected into 4 ml of 0.1% glutaraldehyde buffer for fixation for 15 min. 5. Prior to FCM, the suspension is washed with PBS. Suspend with gentle shaking.
4. Slowly inject the suspension into 4 ml of 0.1% glutaraldehyde buffer for 15 min.
5. Before FCM, wash off the fixative with PBS, add 0.5 ml of 0.01% Peyronie's Y working solution, stain the precipitate for 30 min, centrifuge for 30 min to remove the staining solution and centrifuge for 5 min at 1500 r/min.
6. Wash the cells once with PBS and centrifuge for 5 min at 1500 r/min. 6. Wash the cells once with PBS, centrifuge at 1500 r/min for 5 min, remove the supernatant, and then suspend the cells with PBS.