Flow cytometry

Developed by Justin Meyers 

Sample: Cell line or primary cells 

Materials 

• Propidium iodide reagent (Sigma P-4170) 10mg make up 1mg/ml solution in DI water and store at 4C 
• Rnase A, (Sigma R-6513), 10mg, store at -20C 

Procedure 

Isolate cells 

1. Harvest cells, want good single cell suspension
2. Count cells 
3. Dispense aliquots of 1 million cells into labeled tubes 
4. Add 5 ml PBS and centrifuge for 5 min at 400 x g. 
5. Re-suspend cell pellet in 300 ul PBS with gentle vortex 

Fix cells 

1. Add 700 ul ice cold ethanol (200 proof), slowly, while continuously vortexing. 
2. Incubate at 4C for 30 min to overnight 
3. Pellet cells at 2000 rpm for 5 min 
4. Re-suspend (vortex gently) cells in 250 ul of PBS 
5. Add 5ul of 10mg/ml Rnase A (final concentration being 0.2-0.5 mg/ml) 
6. Incubate at 37C for one hour 
7. Add 10ul of the 1mg/ml PI solution (final concentration is 10ug/ml) 
8. Keep in the dark for at least one hour until analysis 
9. Perform flow cytometry analysis on flow

Developed by Justin Meyers 

Sample: Cell line or primary cells 

Materials 

• Directly conjugated antibodies 
• FACS buffer (DPBS + 5% FBS)  
• BD Cytofix (BD 554655) or 1% paraformaldehyde 

Procedure 

Isolate cells 

1. Harvest cells, want good single cell suspension 
2. Count cells 
3. Dispense aliquots of 1 million cells into labeled tubes 
4. Add 5 ml FACS Buffer and centrifuge for 5 min at 1300 RPM 
5. Re-suspend cell pellet in 100ul FACS Buffer with gentle vortex 

Fix cells 

1. Add appropriate amount of antibody 
2. Vortex gently 
3. Incubate at 4C for 30 min 
4. Add 4 ml of FACS Buffer 
5. Pellet cells at 1300 rpm for 5 min 
6. Re-suspend cells in 500 ul of FACS Buffer 
7. Keep cells on ice up to 1 hour before analysis* 
8. Analyze cells on flow cytometer 

*If cells cannot be analyzed within one hour, fix the cells in 0.5 ml of 1X BD Cytofix or 1% paraformaldehyde and store at 4C for analysis the following day 

Developed by Justin Meyers 

Sample: Cell line or primary cells 

Materials 

• Cells stained with antibody 
• BD Cytofix (BD 554655) 
• D-PBS 
• FBS 

Procedure 

Procedure for fixing cells with BD Cytofix™ 

1. Pellet 106 cells by centrifugation (250 – 300 x g) and carefully remove supernatant 
2. Make up 1X of fixation buffer by adding 5 ml of Cytofix (BD554655) to 10 ml of DPBS 
3. Add either 300μl (for microwell plates) or 500 μl (for tubes) aliquots of 1X fixation buffer to each cell pellet and resuspend the cells by either pipetting or vortexing. 
4. Incubate the cells with fixation buffer for 15 to 30 min at 4°C. (Cell aggregation can be avoided by vortexing prior to the addition of the fixation buffer.) 
5. Fixed cells should be washed and suspended in a buffer that contains protein. (DPBS + 5% FBS) for longer term storage. They can be left in the fixative for up to two days. 

Store the fixed cells at 4°C (protected from light). 

It is recommended that fixed cell samples be read as soon as possible, i.e., within one week.