I work in a cancer lab and have been trying to isolate and quantify platelets without the use of any advanced hematological instruments. I don't need pure platelets, so I've just been generating platelet-rich plasma and trying to get a rough count using a neubauer chamber. With this method though it's incredibly difficult to figure out what I'm looking at under the microscope, and counting platelets has been impossible. Does anyone have experience with this?
Current PRP isolation protocol:
1. Collect blood from mice after CO2 euthanasia via the posterior vena cava, collect into sodium citrate (1:9)
2. Centrifuge whole blood @300g for 20min
3. Collect upper 2/3 plasma fraction
4. Centrifuge fraction @800g for 15min
5. Resuspend in PIPES buffer, pH 7.4
6. Dilute 1:20 and add 10uL to Neubauer chamber
7. Allow cells to settle in moist environment for 15min, try to count
Dear Ma'am Savannah Free please read these articles, they might be helpful for your study.
You can estimate the platelet count by quickly methods with a blood smear microscopy at 100× magnification. At this magnification, the platelet count/µL of blood can be approximated by multiplying the number of platelets seen in one microscope field by 15,000.
Significate formula which actually co relate with real count of Neubauer chambers & hemocytometer
https://www.sciencedirect.com/topics/immunology-and-microbiology/platelet-count
Using 1% ammonium oxalate as diluent
Article Validation of a new method for estimating low platelet count...
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