Detection of platelet-associated antibodies by flow cytometry
Detection of platelet-associated antibodies by flow cytometry
An immunoglobulin that can pass through the placenta and exist on the surface of platelets is called platelet-associated antibody (PA-IgG). This experiment is from "Color Atlas of Practical Flow Cytometry", edited by Shukui Wang and Zhenying Zhou.
Operation method
Detection of platelet-associated antibodies by flow cytometry
Principle
An immunoglobulin that can pass through the placenta to be present on the surface of platelets is known as platelet-associated antibody (PA-IgG).
Materials and Instruments
Sheep F(ab+)2 2 IgG-PE Antibody Blood Sample Move I. Reagents For more product details, please visit Aladdin Scientific website.
Wash buffer
Flow cytometer
Wash buffer (PBS/EDTA/BSA), PBS buffer with 10 mmol/L EDTA and 0.5% BSA.
Antibody: sheep F( ab+ ) 2 anti-human IgG (Fc Sp)-R-PE (H10104, Caltag for polyclonal antibody).
Negative control: sheep F( ab+ ) 2 2 IgG-PE (11304, Caltag).
II. Samples
1 blood sample from a normal person with platelet values in the normal range, exact number unknown.
Patient blood samples 2 samples, 1 month old twins patient 1 and patient 2, both with anemic platelets, exact number not known.
Procedure
1. Blood was collected in EDTA anticoagulant-containing blood collection tubes. 2 ml of each of the 3 blood samples was added to 3 flow-through standard tubes.
2. Centrifugation was performed at 500 r/min, and the supernatant (platelet-rich plasma) was transferred to the other 3 flow-through standard tubes.
3. Centrifugation was performed at 2500 r/min for 3 min. The plasma was aspirated, and platelets were resuspended in a volume of washing buffer equal to that of the plasma. If necessary, mix with a vortex mixer.
4. Wash the platelets three times with wash buffer, centrifuging at 2500 r/min for 3 min each time.
5. Resuspend the platelets in the appropriate amount of wash buffer, and determine the platelet concentration using a hemocytometer with platelet counts of 2000/ul in normocholesterolemic samples, 6000/ul in Patient 1, and 7000/ul in Patient 2 (diluted concentration). The platelet count for Patient 1 is 6000/ul and that for Patient 2 is 7000/ul (diluted concentration).
6. Each flow standard tube containing resuspended platelets is divided into two tubes. tube A is the assay tube and tube B is the negative control tube, for a total of 6 tubes. Normal human blood samples of 250 ul were taken from each of the 2 flow standard tubes (tube A and tube B) with a total platelet count of 5x106. 80 ul of patient 1's blood was taken from each of the 2 flow standard tubes (tube A and tube B) with a total platelet count of 4. 8x106. 70 ul of patient 2's blood was taken from each of the 2 flow standard tubes (tube A and tube B) with a total platelet count of 4.9x106. . The total platelet count of each tube was 4. 9x106.
7. Add 5 ul of goat F( ab+ ) 2 anti-human IgG (Fc Sp) to each of the above 6 flow-through standard tubes, and add 5 ul of goat F( ab+ ) 2 IgG to each of the B tubes.
8. Incubate the blood sample at room temperature, avoiding light for 15 min, and then wash with washing buffer once more as in step 4.
9. Remove the supernatant and resuspend it in 0.5ml of washing buffer, and analyze the blood sample in the machine. 