Experimental Procedure

Sample Preparation

To perform flow cytometry,it is important to have the sample in the form of a single-cell suspension.However,for adherent cells or solid tissues,it becomes necessary to prepare a single-cell suspension prior to flow cytometry staining. Common commonly methods for used ample achieving this include enzymatic digestion or mechanical tissue dissociation.The following are processing methods:

Suspension Cells
  • 1Gently pipette up and down to mix the suspension cells.
  • 2Collect the cells in a 15 mL or 50 mL centrifuge tube (depending on the cell quantity).
Adherent Cells
  • 1Discard the culture medium and wash the cells with 10 mL of 1×PBS, pH 7.4. Then, add 3 mL of 0.05% Trypsin-EDTA solution at room temperature and incubate for 3 minutes. Observe under a microscope for the detachment of a large number of cells.
  • 2Terminate the reaction with 15 mL of culture medium and gently pipette up and down to wash the adherent cells. Collect the cells in a 15 mL or 50 mL centrifuge tube (depending on the cell quantity).
Lymphoid Tissue

Spleen Tissue

  • 1Remove the spleen and place it in a 10 cm dish containing 20 mL of RPMI1640 medium supplemented with 1×P/S.
  • 2Place the spleen in a 75 μm cell strainer and use a pestle to grind the spleen against the strainer, ensuring movement of the strainer while grinding. Rinse the cells off the strainer with medium to disperse them in the medium.
  • 3Transfer the medium into a 50 mL centrifuge tube. Rinse the dish with fresh medium and transfer it to the centrifuge tube as well, keeping the total volume of medium not exceeding 30 mL.
  • 4Red Blood Cell (RBC) Lysis: Centrifuge at room temperature at 400 g for 5 minutes, discard the supernatant, and keep the cells. Add 13 mL of room temperature RBC lysis solution to lyse the red blood cells. Gently pipette to disperse cell clumps and incubate for 1 minute. Add 37 mL of basic culture medium and mix well to stop the RBC lysis.
  • 5Centrifuge at room temperature at 400 g for 5 minutes, discard the supernatant, and keep the cells. Add 40 mL of basic culture medium at room temperature to resuspend the cells and gently pipette to disperse cell clumps, completing the first wash.
  • 6Centrifuge at room temperature at 400 g for 5 minutes, discard the supernatant, and keep the cells. Add 20 mL of 20% FBS culture medium at room temperature to resuspend the cells and gently pipette to disperse cell clumps. Pass the resuspended cells through a cell strainer to remove clumped cells.
  • 7Centrifuge at room temperature at 400 g for 5 minutes and discard the supernatant.
  • 8Add 20 mL of complete culture medium (RPMI1640 + 10% FBS + 1×P/S) to resuspend the cells and prepare for cell counting.

Thymus Tissue

  • 1Remove the thymus and immerse it in a 10 cm culture dish containing 10 mL of serum-free medium with 1% Penicillin-Streptomycin Solution.
  • 2Place the thymus in a 200-mesh sieve and gently grind it with a tissue grinder until no obvious clumps are present.
  • 3Rinse the sieve with 15 mL of PBS, collect the rinse solution in a 15 mL centrifuge tube, centrifuge at 300 g for 5 minutes, and discard the supernatant.
  • 4Resuspend the thymus cells in complete culture medium, pass the suspension through a 200-mesh sieve, centrifuge at 300 g for 5 minutes, discard the supernatant, and resuspend the thymus cells in complete culture medium, adjusting the cell concentration to 1×107/mL.

Staining Process
Cell Surface Staining
  • 1Cell Counting: Collect the cells in a 50 mL centrifuge tube and count them. Centrifuge at 500 g for 4 minutes, discard the supernatant.
  • 2Preparation of Single Cell Suspension: Resuspend the collected cells in 1 mL of 0.5% BSA/PBS solution. Centrifuge at 400 g for 3 minutes, discard the supernatant, and repeat this step twice. Resuspend the cells in 0.5% BSA/PBS solution and distribute them into a 96-well plate, adding 50 μL of cell suspension per well.
  • 3Blocking (optional): Depending on the staining protocol and cell species, incubate with 10% goat serum/10% mouse serum/commercial blocking reagent at room temperature for 30-60 minutes to block non-specific binding mediated by Fc receptors.
  • 4Incubation with Primary Antibody: Add 50 μL of primary antibody dilution to each well. Shake gently and incubate at room temperature for 20 minutes.
  • 5Wash: Centrifuge at 400 g for 5 minutes, discard the supernatant.
  • 6Incubation with Secondary Antibody:If using a fluorescent dye-conjugated secondary antibody, add 100 μL of fluorescent secondary antibody to each well. Resuspend gently in the dark and incubate at room temperature for 20 minutes. If using directly-labeled antibodies, skip to step 9.
  • 7Centrifuge at 400 g for 5 minutes, discard the supernatant.
  • 8Fixation (optional): If not immediately analyzing the stained cells, resuspend the cells in 100 μL of 4% paraformaldehyde, incubate at room temperature for 10 minutes, and wash twice with 0.5% BSA/PBS solution.
  • 9Wash: Add 200 μL of 0.5% BSA/PBS solution, centrifuge at 400 g for 5 minutes, discard the supernatant, and repeat this step twice.
  • 10Resuspend the cells in 200 μL of 0.5% BSA/PBS solution and analyze them using the appropriate instrument.
Intracellular Staining

Formaldehyde/Saponin Method (Cytoplasmic Proteins)

  • 1Sample Preparation: Collect the cells to be tested, count and assess viability. Then, resuspend the cells in 1 mL of 0.5% BSA/PBS solution, wash twice, centrifuge at 400 g for 3 minutes, and discard the supernatant.
  • 2Viability Staining: Use a dilution of L/D dye, stain at room temperature in the dark for 15 minutes, centrifuge and discard the supernatant, then wash twice with 1 mL of 0.5% BSA/PBS solution.
  • 3Cell Fixation: Resuspend the cells in 4% paraformaldehyde fixation solution, rotate and incubate in the dark for 15 minutes, followed by one wash with 0.5% BSA/PBS solution.
  • 4Cell Permeabilization: Prepare a permeabilization solution by adding 0.1% saponin to 0.5% BSA/PBS solution. Resuspend the cells in this permeabilization solution, incubate in the dark for 1 hour, then centrifuge and discard the supernatant. (All buffers used after this step should contain 0.1% saponin since saponin permeabilization is reversible.)
  • 5Incubation with Primary Antibody: Resuspend the cells in 0.5% BSA/PBS solution containing 0.1% saponin, distribute them into a 96-well plate (50 μL per well, with a cell count of ≤1E6 per well), and add the primary antibody dilution (diluted in 0.5% BSA/PBS solution containing 0.1% saponin), 50 μL per well. Mix well. Place the 96-well plate on a shaker, incubate at room temperature in the dark for 30 minutes.
  • 6Wash to Remove Unbound Primary Antibody: After incubation, centrifuge at 400 g for 5 minutes and discard the supernatant. Wash each well with 200 μL of 0.5% BSA/PBS solution containing 0.1% saponin, centrifuge at 400 g for 5 minutes, and discard the supernatant.
  • 7Incubation with Secondary Antibody: If the primary antibody is not directly labeled with a fluorescent dye, add the appropriate fluorescent secondary antibody dilution (diluted in 0.5% BSA/PBS solution containing 0.1% saponin) for the corresponding species, 100 μL per well. Incubate in the dark at room temperature on a shaker for 30 minutes. If the primary antibody is already labeled with a fluorescent dye, proceed to step 9 after repeating step 6.
  • 8Wash to Remove Unbound Secondary Antibody: After incubation, centrifuge at 400 g for 5 minutes and discard the supernatant. Wash each well twice with 200 μL of 0.5% BSA/PBS solution containing 0.1% saponin, centrifuge at 400 g for 5 minutes, and discard the supernatant.
  • 9Resuspend for Analysis: Resuspend the cells in 100-200 μL of 0.5% BSA/PBS solution containing 0.1% saponin and analyze using the appropriate instrument.

Formaldehyde/Triton Method (Nuclear Proteins)

  • 1Sample Preparation: Collect the cells to be tested, count and assess viability. Then, resuspend the cells in 1 mL of 0.5% BSA/PBS solution, wash twice, centrifuge at 400 g for 3 minutes, and discard the supernatant.
  • 2Viability Staining: Use a dilution of L/D dye, stain at room temperature in the dark for 15 minutes, centrifuge and discard the supernatant, then wash twice with 1 mL of 0.5% BSA/PBS solution.
  • 3Cell Fixation: Resuspend the cells in 4% paraformaldehyde fixation solution, rotate and incubate in the dark for 15 minutes, followed by one wash with 0.5% BSA/PBS solution.
  • 4Cell Permeabilization and Blocking: Resuspend the cells in Triton X-100 permeabilization solution, rotate and incubate in the dark for 15 minutes, centrifuge and discard the supernatant, then resuspend in Fc receptor blocking solution, incubate for 1 hour, and centrifuge and discard the supernatant.
  • 5Incubation with Primary Antibody: Resuspend the cells in 0.5% BSA/PBS solution, distribute them into a 96-well plate (50 μL per well, with a cell count of ≤1E6 per well), and add the primary antibody dilution (diluted in 0.5% BSA/PBS solution), 50 μL per well. Mix well. Place the 96-well plate on a shaker, incubate at room temperature in the dark for 30 minutes.
  • 6Wash to Remove Unbound Primary Antibody: After incubation, centrifuge at 400 g for 5 minutes and discard the supernatant. Wash each well with 200 μL of 0.5% BSA/PBS solution, centrifuge at 400 g for 5 minutes, and discard the supernatant.
  • 7Incubation with Secondary Antibody: If the primary antibody is not directly labeled with a fluorescent dye, add the appropriate fluorescent secondary antibody dilution for the corresponding species, 100 μL per well. Incubate in the dark at room temperature on a shaker for 30 minutes. If the primary antibody is already labeled with a fluorescent dye, proceed to step 9 after repeating step 6.
  • 8Wash to Remove Unbound Secondary Antibody: After incubation, centrifuge at 400 g for 5 minutes and discard the supernatant. Wash each well twice with 200 μL of 0.5% BSA/PBS solution, centrifuge at 400 g for 5 minutes, and discard the supernatant.
  • 9Resuspend for Analysis: Resuspend the cells in 100-200 μL of 0.5% BSA/PBS solution and analyze using the appropriate instrument.

Learn More

ABflo™Flow Cytometry Antibodies Common Markers for Immunophenotyping Online Resources