Begin by transferring previously thawed pancreatic tumor cell suspension to a two-milliliter microcentrifuge tube and add PBS to reach a final volume of two milliliters. Then, use a hemocytometer to evaluate the quantity and quality of the cells. Once the cell pellet is obtained through centrifugation, using progressively smaller pipette tips, carefully decant the supernatant to minimize pellet distribution while maximizing the fluid decanted.
Prepare 1X cell lysis buffer, then add 100 microliters of it to 900 microliters of previously prepared cell lysis dilution buffer to obtain 0.1X cell lysis buffer. Transfer 100 microliters of chilled 0.1X cell lysis buffer to the cell pellet and gently pipette five times to ensure complete resuspension. After incubation on ice for three minutes, add one milliliter of chilled wash buffer to the resuspended pellet and gently pipette it five times.
Following centrifugation, discard the supernatant as demonstrated, and repeat this washing process two more times. Load trypan blue-stained suspension into a hemocytometer to count the number of nuclei. Resuspend the nuclei pellet in 1X nuclei buffer based on goal-targeted nuclei recovery.
Carefully pass the resuspended nuclei through a 40-micrometer pipette tip cell strainer, and then maintain it on ice. Now recount the nuclei. The nuclei obtained using this method exhibited appropriate size and shape.
Occasional mild stippling of the nuclear envelope can be observed and should be monitored at the final nuclear evaluation via hemocytometer.
