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A protocol of Peripheral Blood Leukocyte Culture:
- Collect 2ml blood in heparin tube, aseptically.
- Wipe all the instruments and equipment with alcohol and UV-sterilize it.
- In a 15ml falcon tube, add 7ml RPMI-1640 complete media, close the bottle and cover it with parafilm.
- Add 100mg/ml 10μl and 20μl of penicillin and streptomycin antibiotics to culture, respectively.
- Add 200mM of 100µL L-glutamine and 100µL of phytohemagglutinin M to the culture one after another.
- add 0.7ml to 1ml of blood to the falcon tube, carefully. Fix the culture by inverting tubes some time, gently.
- In the CO2 incubator, incubate culture at 37℃ for 70 to 72 hours. Put tubes at a 45-degree angle to grow cells well.
- At the 70th hour add 100µL of 0.2% colcemid to the culture
- Incubate the culture for two more hours, meanwhile, prepare the fixative for harvesting.
- First, centrifuge the culture tubes at 3500rpm for 8 to 10 minutes at room temperature
- Discard the supernatant and add pre-warmed 0.56% KCl to the culture. Add hypo to up to 8ml mark, roughly.
- Incubate pellets in the prewarmed water bath at 37℃ for 15 to 17 minutes. Immediately, after the hypotonic treatment, add chilled fixative. Mix well till it homogenized.
- Repeat centrifugation at 2500rpm to 3500rpm for 8 to 10 minutes. To the pellet add 8ml chilled fixative. Centrifuge it properly again. Repeat, centrifugation, and fixative step until clear white pellet appears.
- In the last step, store the pellets in 2ml fixative at 4℃