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℃