Isolation of mouse footpad cells for immunophenotyping

Hi Kar Wei,

Thanks for your inquire. Mouse footpads used for cell isolation and cytokine/chemokine quantification were not from the same animals, those were independent procedures as the protocols used differ. For a detailed description of the protocol used to obtain footpad lysates for cytokine/chemokine quantification, please refer to the Materials and Methods section entitled "Cytokine/chemokine quantification by multiplexed bead-based immunoassays" in the original article. Below is the detailed protocol for cell isolation from mouse footpads. We hope it helps you. 

Mouse footpad cell isolation for immunophenotyping: 

Materials:

• 6-well culture plates.
• 40µm cell strainers.
• 40mm Petri dishes.
• 1ml syringes.
• 50ml centrifuge tubes.
• 5ml round bottom polystyrene test tubes.
• 10x Flow Cytometry Mouse Lysis Buffer (R&D systems, Cat. # FC003).
• Percoll® (Sigma-Aldrich, Cat. # P1644)
• Complemented RPMI (cRPMI, recipe below).
• Digestion medium (recipe below).
• Serological pipettes. 
• Scissors and toothed forceps.

Complemented RPMI (for 500ml):

• 450ml RPMI-1640 +25mM HEPES + L-Glutamine (HyClone™ Cat. # SH30255.01)
• 50ml Fetal Bovine Serum Characterized (FBS) (HyClone^TM, Cat. # SH30071.03)              

Digestion medium (for 50ml):

• 1ml 50X Dispase II (100U/ml) (Gibco™, Cat. # 17105-041)                            
• 1ml 50X Collagenase IV (10mg/ml) (Sigma-Aldrich, Cat. # C5138)               
• 250ul 200X DNase I (10mg/ml) (Roche Applied Science, Cat. # 10104159001)
• 47.75 ml cRPMI

Equipment:

• Centrifuge.

Procedure:

• Euthanize mice by CO₂ asphyxiation.
• Cut the footpads right above the ankle and place them each into a 6-well culture plate filled with 4ml of digestion medium (see recipe).
• Deskin the footpads and mince them using toothed forceps. Remove as much flesh as possible from the bones. Incubate minced footpads at 37ºC for 3 hours, 5% CO₂ on a shaker (~100 rpm/min). 
• Place a 40µm cell strainer into an empty 40mm Petri dish and add 5ml of cRPMI. Transfer digested mix to the cell strainer to remove undigested tissue. Using the flat base of a 1ml syringe plunger, smash undigested tissues against the strainer using circular motion (this step helps maximize the cells recovered from undigested tissue). 
• Place the used cell strainer on a 50ml centrifuge tube. Pour the medium containing the cells from the Petri dish into the centrifuge tube (passing through the cell strainer). Rinse the Petri dish with 5 ml of cRPMI and pour it into the centrifuge tube, passing through the cell strainer again.
• Discard cell strainer and cap the centrifuge tube. Spin down the cells for 5 minutes, 1500rpm. Discard the supernatant.
• Add 4ml of sterile 1x Flow Cytometry Mouse Lysis Buffer, mix well and incubate at room temperature (RT) for 5 minutes (the medium should turn red as hemolysis occurs).
• Top up with cRMPI medium to 10 ml, and spin for 5 minutes, 1500rpm. Discard the supernatant.
• Resuspend the cell pellet in 1ml of cRPMI. 
• To get rid of cell debris by density centrifugation, prepare 5ml round bottom polystyrene test tubes containing 2ml of 35% v/v Percoll solution in cRPMI (keep the tubes with Percoll cold at 4ºC). Carefully lay cell suspension (from previous step) on top of the Percoll solution. Use a pre-chilled centrifuge (4ºC) and spin tubes at 2,400 rpm for 20 (acceleration/deacceleration=1 or minimum). 
• Carefully remove cell debris from the interphase layer using a micropipette. Proceed to remove as much supernatant as possible without disturbing the cell pellet. Resuspend cells with 2ml of cRPMI. Spin tubes for 5 minutes, 1500rpm. Discard the supernatant.
• Resuspend in 1ml of cRPMI. 
• Proceed with downstream procedures (cell counting, immunostaining, etc).