Editing Isolation of murine splenocytes (section)

=== Protocol A ===

Set-Up

#Clean dissection stage with 70% ethanol.
#Add ethanol to the beaker and place ends of scissors and forceps into the beaker to sterilize.
#Add 8-10mL of DMEM-10 to the petri dish.
#Place the cell strainer into the dish with the DMEM-10.

Procedure

#Wet fur on left side of sacrificed mouse using 70% ethanol.
#Cut out the spleen.
##Cut away the fur along the left side of the mouse, about half-way between the front and back legs.
##Cut open the body cavity.
##Remove the spleen using the forceps (the spleen is the color of a kidney bean; it is longer and flatter than the kidney).
#Place the spleen into the cell strainer. Using the plunger end of the syringe, mash the spleen through the cell strainer into the petri dish.
#Rinse the cell strainer with 5mL DMEM-10. Discard the strainer.
#Transfer the suspended cells to a 15mL conical.
#Spin cells at 800xg for 3 minutes.
#Discard supernatant and resuspend pellet in 1mL ACK lysis buffer. Incubate at RT for 5-10 minutes. Add 9mL DMEM-10 and spin as before.
#Discard supernatant and resuspend pellet in 3mL DMEM-10, discarding any dead cell mass.
#Count cells (dilute 10μL cell suspension in trypan blue, and count with hemcytometer).