It is well known that good sample preparation is half of the success of an experiment. Single cells are a prerequisite for the analysis and detection of cells by flow cytometry. Therefore, when performing flow experiments, the preparation of qualified and dispersed single-cell suspensions is an important part of the flow cytometry sample preparation technique, which requires the tissue to be dispersed into single cells, but also maintains the intrinsic properties and biological characteristics of the cells. Today, we list a few common flow tissue sample single cell suspension preparation solutions to help flow experiments.
Spleen
Spleen single cell suspension preparation procedure:
1 Remove the mouse spleen and soak it in clean PBS solution.
2 Place the spleen in a 200 mesh sieve and gently grind with a tissue grinder stick until there are no visible red clumps.
3 Rinse the sieve with 15 mL of PBS and collect the rinse solution in a 15 mL centrifuge tube and centrifuge at 300 g for 5 min, discarding the supernatant.
4 Resuspend the cells by adding 2 mL of 1× erythrocyte lysate, and after lysis for 2~3 min at room temperature, immediately add 10 mL of PBS, centrifuge at 300 g for 5 min, and discard the supernatant.
5 Resuspend splenocytes with Cell Staining Buffer, filter the cell suspension through a 200-mesh sieve and count the cells, and adjust the cell concentration to 1×107 cells/mL.
Bone Marrow
Procedure for preparation of single-cell suspension of bone marrow samples:
1 Carefully pinch the abdominal skin between the two hip joints of the mouse with ophthalmic forceps, and ophthalmic scissors carefully cut and separate the skin of the lower limbs. Cut the skin downward at the ankle and upward at the hip joint to free both hind limbs of the mouse.
2 Carefully peel back the muscles and cut the femurs (femur) and tibias (tibia) separately, clipping the cartilage at both ends to expose the red marrow cavity. Note that as much of the marrow cavity as possible should be preserved during this procedure.
3 Using a 1 mL sterile syringe, aspirate 1 mL of PBS solution and gently insert it into the bone marrow cavity to flush the marrow cavity to obtain bone marrow. Repeat 2 to 3 times to flush out the majority of cells. After flushing, gently blow the cells with a pipette to disperse the cell mass.
4 Filter the rinsate through a 200-mesh filter, collect the filtrate in a 15-mL centrifuge tube, centrifuge at 300 g for 5 min, and discard the supernatant.
5 Resuspend the cells with cell staining buffer and count, adjust the cell concentration to 1×107 cells/mL.
Thymus
Thymus single cell suspension preparation procedure:
1 Remove the thymus and soak it in clean PBS solution.
2 Place the thymus in a 200 mesh sieve and gently grind it with a tissue grinder stick until there are no visible lumps.
3 Rinse the screen with 15 mL of PBS and collect the rinsate in a 15 mL centrifuge tube and centrifuge at 300 g for 5 min, discarding the supernatant.
4 Resuspend thymocytes with cell staining buffer and count, adjusting the cell concentration to 1 × 107 cells/mL.
Lymph nodes
Lymph node single cell suspension preparation procedure:
1 Remove lymph nodes and soak in clean PBS solution.
2 Aspirate the culture solution with a 2.5 mL sterile syringe, hold the lymph node with forceps in the left hand, hold the syringe in the right hand, and carefully insert it into the lymph node and blow it out until the lymph node cells are completely blown out, and observe that only white connective and adipose tissues remain.
3 The blown thymocytes were filtered through a 200-mesh sieve, collected in a 15 mL centrifuge tube, centrifuged at 300 g for 5 min, and the supernatant was discarded.
4 Thymocytes were resuspended with cell staining buffer, counted, and the cell concentration was adjusted to 1 × 107 cells/mL.
The analysis of various parameters of cells by flow cytometry must be based on single cells, therefore, when dealing with different tissues, the preparation of single-cell suspensions with less damage to cells and higher yields should be used as much as possible to maximize the preservation of the original characteristics of cells.
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