Cell Preparation for Flow Cytometry

Cell for flow cytometry analysis are usually derived from four main sources: adherent or suspension cultured cell lines, cryopreserved samples, whole blood and solid tissues such as bone marrow, spleen, and thymus. The most straight forward samples for flow cytometry are non-adherent cells. Here we describe several different methods that provide a general procedure for the preparation of cell suspensions. A certain level of technical skill and immunological knowledge are required for the successful design and implementation of these techniques. The following are just guidelines and may need to be adjusted or optimized for particular applications. 

Materials

• Phosphate buffered saline containing 1% bovine serum albumin (PBS/BSA)
• 0.25% Trypsin
• Culture medium with 10% FBS
• Histopaque or Ficoll
• Ammonium chloride lysis buffer (0.16 M ammonium chloride, 0.17 M Tris, pH 7.2)

Preparation of cells stored in liquid nitrogen

• Thaw the cryotubes rapidly in a water bath set at 37°C.
• Resuspend cells in cold PBS/BSA and transfer them to a 15 mL centrifuge tube.
• Centrifuge at 300-400 g for 5-10 min at 4°C.
• Discard supernatant and resuspend pellet in a suitable volume of cold PBS/BSA.
  Note: Higher viability can be obtained by allowing the cells to recover in culture media overnight.

Preparation of tissue culture cell lines in suspension

• Decant cells from tissue culture vessel/flask into 15 mL centrifuge tube(s).
• Centrifuge at 300-400 g for 5-10 min at room temperature.
• Discard supernatant and resuspend pellet in 10 mL of PBS/BSA and repeat previous step.
• Discard supernatant and resuspend to a minimum concentration of 1 x 10⁷ cells/mL in cold PBS/BSA.

Preparation of adherent tissue culture cell lines

• Remove the culture medium and rinse the cell monolayers with sterile, room temperature PBS.
• Slowly add 0.25% Trypsin to just cover the cell monolayer.
• Incubate at 37°C for 5-10 minutes (depending on the cell type).
• Neutralize the reaction by adding growth medium and resuspend the cells by gently pipetting.
• Continue as with suspension culture cell preparation protocol.
  Note: Epitopes may be cleaved when using the enzymatic digestion method. Cells can also be obtained by gently scraping them into culture media.

Preparation of peritoneal macrophages, bone marrow, thymus and spleen cells

• Prepare a single cell suspension from relevant tissue. Keep cells on ice to minimize cell death, which can lead to cell aggregation.
• Pellet down the cells at 300-400 g for 5 min at 4°C.
• Discard supernatant and resuspend pellet in 10 mL ammonium chloride lysis buffer.
• Mix and incubate for 5 min at 4°C. 
• Pellet down the cells at 300-400 g for 5 min at 4°C.
• Discard the lysis buffer and wash once with cold PBS/BSA.
• Perform a cell count and adjust suspension to a final concentration of 0.7-1.2 x 10⁷/mL if necessary.

Preparation of human peripheral blood mononuclear cells (PBMCs)

• Dilute whole blood with equal volumes of room temperature PBS/BSA (for example, add 3 mL of PBS/BSA to 3 mL of blood).
• Carefully overlay the diluted blood onto an equal volume of gradient media in a 15 mL centrifuge tube.
• Centrifuge at 300-400 g for 30 min at room temperature. The centrifuge brakes should be turned off.
• Harvest the PBMCs from the thin interface between the upper plasma layer and lower medium layer using a pipet.
• Resuspend the cells in PBS/BSA and centrifuge at 300-400 g for 5 min at room temperature.
• Discard supernatant and resuspend pellet to a final concentration of at least 1 x 10⁷ cells/mL with cold PBS/BSA.

For research use only. Not for any other purpose.