Flow cytometry and cell sorting

Protocol for PMA+ I stimulation

• Plate 2-3X10⁶ cells /well
• Stimulate with PMA working concentration 25ng/ml + Ionomycin 0.5 µg/ml
• Our stock concertation is 1mg/ml for both PMA + Ionomycin 
• Usually I make 2X mixture from both ( 1µl of PMA in 20ml of CTM media)
• Take required amount (for example 10ml) from PMA in media and add 10 µl of Ionomycin this makes (PMA+I 2X) mixture.
• Wash cells with CTM media (S-MEM media +10%FBS+antibiotics and amino acids)) and spin down at 1500 rpm/5min
• Pour supernatant and assuming there is 10-20 µl left in each well add 30 µl from PMA+I (2X mix)
• Add equivalent amount 30 µl from CTM to each well to make (1X)
• Incubate cells at 37C for 5 hrs
• Make 5X of Monensin + brefeldin A in (PMA+I 2X) mix ( for example 1ml of PMA+I 2X mix +  5 µl of Monensin + brefeldin (1000X)
• After 1 hr of stimulation add 20 µl from 5X PMA+I +Monensin + brefeldin into each stimulated well
• (* you can also use 1X PMA+I instead of 2X by diluting you 2X mix before adding to wells- need to add same amount of media in this case you add 10ml of media to 10ml of 2X PMA+I in media)
• (* The reason we make 2X mix is to save the amount of media used every time)
• For unstimulated well add 80 µl of 1X of Monensin + brefeldin to each well 
• After 5 hrs ( total time) incubation at 37C, wash cells with 100 µl FACS buffer
• Spin down 1500rpm/5min
• Follow with regular surface staining and fixation/ Intercellular staining 
• Spin down All antibodies (surface and intracellular) before usage 
• For fixation use 2%Formaldehyde methanol free (45min-1hr) at 4C and wash with 1X (eBio perm wash #008-3333-56)
• All intracellular staining is in eBio perm wash for 1hr at 4C.
• Wash with eBio perm wash and then re-suspend the cell in flow buffer.
• Run samples for flow-cytometry analysis
• Only one clone in PE that seems to work for IL-10 intracellular staining under PMA and ionomycin stimulation.

PE anti-mouse IL-10 Antibody Biolegend  catalog# 505008

• Note: the PMA+I stimulation and % of fixation would affect the expression of Foxp3. So, we use 2%PFA instead of 4%PFA and we stimulate with PMA working concentration 25ng/ml + Ionomycin 0.5 µg/ml instead of working concentration 50ng/ml + Ionomycin 1µg/ml (which is the usual concentration for PMA and I stimulation in literature) to maintain the expression of Foxp3.