Flow cytometry sample preparation techniques

Single cells are a prerequisite for the analysis and detection of cells by flow cytometry. In the application of flow cytometry, the preparation of qualified dispersed single cells is an important part of the flow cytometry sample preparation technique. It requires both the dispersion of tissues into single cells and the maintenance of the inherent biochemical composition and biological properties of the cells.

Basic principles of sample preparation for flow cytometry
1. Ensure that all kinds of liquid and suspension cell samples are fresh, as soon as possible to complete the sample preparation and detection. 2.
2. For different cell samples, carry out appropriate washing, enzymatic digestion or EDTA treatment, in order to remove impurities, so that the adherent cells are separated from each other to form a single-cell state. 3.
3. For fresh solid tumor tissues, enzymatic digestion, mechanical dispersion, or chemical dispersion may be used to obtain a sufficient number of single-cell suspensions. 4.
4. For paraffin-embedded tissues, the single-cell suspension should be prepared by the method described above after being cut into several 40-50 μm thick wax slices and deparaffinized to water by xylene.
5. The number of cells in the single-cell suspension should not be less than 107 cells/mL.

flow cytometry experimental operation is roughly divided into the following five steps
1. sampling: take surgical or biopsy tissue must be representative, such as taking surgical tumor tissue, must take the tumor cell growth site; tissue and other specimens must be taken after the sample to maintain the freshness of the samples; generally within one hour at room temperature to deal with the samples or timely preservation of the tissue with a fixative or low temperature.
2. Perform fluorescent staining of cells for biochemicals to be measured.
3. samples are acquired, assayed, and stored according to the software program provided by the manufacturer.
4. Analyze the results qualitatively and quantitatively according to the software program provided.
5. analyze and evaluate the biological and medical significance of the test results.

Preparation of peripheral blood specimens
a) Routine blood sample preparation
1. Peripheral blood was collected and anticoagulated with heparin; it was stored at room temperature (25°C) and processed within 6 h after collection;
2. Erythrocyte lysate (ACK buffer) was removed and returned to room temperature for use;
3. After 200 ul of peripheral blood was added to the flow-through tube, 3 mL of ACK buffer was added and incubated at room temperature for 3~5 min;
4. Add 10 mL of cell staining buffer (or PBS containing 1% BSA) to terminate erythrocyte lysis;
5. centrifuge the cell suspension at 300 g for 5 min and discard the supernatant;
6. repeat the wash with cell staining buffer (or PBS containing 1% BSA) once;
7. adjust the cell concentration to 1x107/mL with cell staining buffer (or PBS containing 1% BSA) and set aside.

b) Peripheral blood single nucleated cell sample preparation
1. 2 mL of peripheral blood was collected, anticoagulated with heparin, and the blood was diluted to 4 mL with saline and mixed well;
2. 15 mL of centrifuge tube, first add an equal volume (4 mL) of lymphocyte isolate;
3. slowly add the diluted peripheral blood along the wall of the tube to the level of the lymphocyte separation solution, do not use excessive force as this may cause the blood to mix with the separation solution. Be sure to maintain clear stratification;
4. 400 g room temperature centrifugation for 20-30 min (according to the amount of blood samples to determine the centrifugation conditions, it is recommended to feel the conditions in order to achieve the best separation effect); centrifugation of the blood in the tube can be seen clearly divided into four layers, the top layer of plasma layer, the second for the white lymphocyte layer, the third for the transparent separation of the liquid layer, and the bottom for the red blood cell layer;
5. the second layer of lymphocyte layer was carefully sucked out with a pipette and collected into another test tube, washed twice with cell staining buffer (or PBS containing 1% BSA), and centrifuged at 250 g for 10 min each time;
6. Adjust the cell concentration to 1x107/mL with cell staining buffer (or PBS containing 1% BSA) and set aside.

Bone marrow cell sample preparation
a) Routine bone marrow cell sample preparation
1. 0.5 mL of bone marrow fluid was extracted aseptically and anticoagulated with heparin;
2. Erythrocyte lysate (ACK buffer) was removed and returned to room temperature for use;
3. 3 mL of ACK buffer is added to the bone marrow fluid and incubated at room temperature for 3~5 min;
4. 10 mL of cell staining buffer (or PBS with 1% BSA) was added to terminate erythrocyte lysis;
5. centrifuge the cell suspension at 300 g for 5 min and discard the supernatant;
6. repeat the wash with cell staining buffer (or PBS containing 1% BSA) once;
7. adjust the cell concentration to 1x107/mL with cell staining buffer (or PBS containing 1% BSA) and set aside.

b) Bone marrow fluid single nucleated cell sample preparation
1. 0.5 mL of bone marrow fluid is aseptically aspirated;
2. drop the bone marrow fluid specimen into 1 mL of PBS solution containing 1000 U/mL heparin anticoagulant;
3. add PBS diluent to dilute the sample to 10 mL;
4. a 15 mL centrifuge tube is started with 5 mL of Lymphocyte Isolate;
5. 5 mL of diluted bone marrow fluid is slowly pipetted along the wall of the tube and added to the top of the lymphocyte isolate;
6. Centrifuge at 400 g for 20~30 min (according to the amount of bone marrow samples to determine the centrifugation conditions, it is recommended to feel the conditions in order to achieve the best separation effect); after centrifugation, the bone marrow fluid can be seen clearly divided into four layers in the test tube, the upper layer is the PBS dilution layer, the second layer is the white lymphocyte layer, the third layer is the transparent separating liquid layer, and the fourth layer is the red blood cell layer;
7. The second layer of lymphocytes was carefully sucked out with a pipette and collected into another test tube, washed twice with cell staining buffer (or PBS containing 1% BSA), and centrifuged at 300 g for 5 min each time;
8. Adjust the cell concentration to 1x107/mL with cell staining buffer (or PBS containing 1% BSA) and set aside.

Preparation of body fluids, lavage fluids or suspension-cultured cells
1. Collect fresh body fluid, lavage fluid or suspension culture cells. 2;
2. centrifuge at 300 g for 5 min, and collect the cell precipitate;
3. Wash the cells by centrifugation with cell staining buffer (or PBS containing 1% BSA) for 1~2 times;
4. If there are obvious precipitate clots in the suspension, filter the suspension through a 200-300 mesh filter and then wash with cell staining buffer (or PBS containing 1% BSA) for 1-2 times;
5. Adjust the concentration to 1x107/mL with cell staining buffer (or PBS containing 1% BSA).

Preparation of adherent cells
1. Aspirate the supernatant of culture medium and rinse it once with PBS containing no calcium and magnesium ions. 2;
2. Take 6-well plate as an example, add 1 mL of digestive solution (0.1% trypsin) to each well, and digest for 2~5 min at room temperature (25℃) or 37℃; or add 1 mL of EDTA (0.02%, pH7.4) and let it stand on ice for 5~10 min. 3;
3. Observe under the inverted microscope, and find that the cytoplasm is retracted and the gap is enlarged, immediately aspirate the digested liquid and add an equal volume of serum-containing medium to terminate digestion;
4. Aspirate the liquid in the bottle and blow the cells on the wall of the bottle repeatedly. The blowing action should be gentle and try to avoid foam. 5;
5. Transfer to a centrifuge tube and centrifuge at 300 g for 5 min, discard the supernatant. 6;
6. centrifuge with cell staining buffer (or PBS containing 1% BSA) for 1~2 times. 7;
7. Adjust the cell concentration to 1x107/mL with cell staining buffer (or PBS containing 1% BSA).

Preparation of tissue specimens
a) Shearing and grinding method:
1. rinse the tissue with PBS or serum-free medium;
2. place the tissue in a flat dish and cut into small particles (1~2 mm3 ) with ophthalmic shears;
3. Grind into homogenate with syringe needle core;
4. Add appropriate amount of PBS or serum-free medium dropwise and mix by pipetting;
5. Filter through 200~300 mesh filter, centrifuge at 300 g for 5 min, and discard the supernatant;
6. centrifuge with cell staining buffer (or PBS containing 1% BSA) for 1~2 times;
7. Adjust the cell concentration to 1x107/mL with cell staining buffer (or PBS containing 1% BSA).

b) Net rubbing method
1. Tie a 300-mesh nylon net on a small beaker;
2. put the cut tissue on the net, use ophthalmic forceps to gently rub the tissue block, add PBS to rinse while rubbing until the tissue is rubbed out;
3. Collect the cell suspension, centrifuge at 300 g for 5 min, and discard the supernatant. 4;
4. Wash by centrifugation with cell staining buffer (or PBS containing 1% BSA) for 1~2 times. 5;
5. Adjust the cell concentration to 1x107/mL with cell staining buffer (or PBS containing 1% BSA) and set aside.

c) Grinding method
1. cut the tissue into 1~2 mm3 size pieces;
2. Place in a tissue grinder and add 1~2 mL of PBS;
3. Turn the rod and grind until homogenized. 4;
4. Add 10 mL of PBS and rinse the grinder. 5;
5. Collect the cell suspension, filter through 200~300 mesh nylon mesh, centrifuge at 300 g for 5 min, and discard the supernatant;
6. Wash by centrifugation with cell staining buffer (or PBS containing 1% BSA) for 1~2 times;
7. Adjust the cell concentration to 1x107/mL with cell staining buffer (or PBS containing 1% BSA).

Note: 1) The above method is a mechanical method, used to deal with some soft tissues such as thymus, lymph nodes, etc. It is not effective for hard or fibrous tissues, and has some damage to tissue cells.
(2) If the tissue is spleen, it can be treated by grinding method and then do erythrocyte lysis. After centrifugation at 300 g for 5 min and discarding the supernatant, add 3 mL of ACK buffer and incubate at room temperature for 3~5 min; then add 10 mL of cell staining buffer (or PBS containing 1% BSA) to terminate the erythrocyte lysis; the subsequent operation is the same as that of the grinding method.

d) Trypsin digestion:
It is suitable for digesting tissues with less mesenchyme, such as epithelium, liver, kidney, etc. Since calcium ions or serum will inhibit the digestive action of trypsin, the liquid used in this process should be free of these ions or serum. The digestion time is adjusted according to different situations; low temperature, large tissue mass, and low concentration of trypsin result in a long digestion time; conversely, the digestion time is shortened. Trypsin is often mixed with EDTA (0.02%) in equal proportions to improve digestion efficiency.

1. Rinse the tissue block with calcium and magnesium ion free PBS;
2. place the tissue in a flat dish and cut into small pellets (1~2 mm3) with ophthalmic scissors;
3. add 30 times the amount of tissue in protease solution (0.1% trypsin and 0.02% EDTA mixed in equal proportions);
4. Transfer the pipette to a triangular flask and place it in a 37℃ water bath or thermostat to digest for 20~60 min, with shaking every 5~10 min. If the digestion takes longer time, 2/3 of the digested supernatant can be transferred to centrifuge tube every 15 min for ice bath or centrifugation to remove the digested liquid, add serum-containing medium to terminate the digestion, and then replenish new digested liquid into the triangular flask to continue digestion. 5;
5. Filter the digested solution or batch-collected cell suspension through a 200~300 mesh filter, centrifuge at 300 g for 5 min, and discard the supernatant;
6. centrifuge with cell staining buffer (or PBS containing 1% BSA) for 1~2 times;
7. Adjust the cell concentration to 1x107/mL with cell staining buffer (or PBS containing 1% BSA) and set aside.

e) Collagenase digestion:
This method is suitable for isolating fibrous, epithelial and cancerous tissues. Calcium and magnesium ions do not inhibit digestion, so it can be prepared with PBS or serum-containing medium to improve cell viability.

1. Rinse the tissue block with PBS. 2;
2. place the tissue in a flat dish and cut it into small pellets (1~2 mm3) with eye scissors;
3. Add 30 times the amount of tissue in protease solution (0.1~0.3 μg/mL of collagenase);
4. Transfer the pipette to a triangular flask and place the pellet in a 37℃ water bath or a thermostat for 4~48 h. Shake every 5~10 min. Or every 15 min, 2/3 of the digested supernatant can be transferred to a centrifuge tube in an ice bath or centrifugation to remove the digested liquid, add serum-containing medium to terminate the digestion, and then replenish new digested liquid into the triangular flask to continue digestion. 5;
5. Filter the digested solution or batch-collected cell suspension through a 200~300 mesh filter, centrifuge at 300 g for 5 min, and discard the supernatant;
6. centrifuge with cell staining buffer (or PBS containing 1% BSA) for 1~2 times;
7. Adjust the cell concentration to 1x107/mL with cell staining buffer (or PBS containing 1% BSA).

Note: The above are the most common single-cell preparation methods for solid tissue samples. Mechanical methods often cause serious cell damage and low single-cell yield; enzymatic methods and chemical methods (combined use of enzymes and EDTA) are more desirable for dispersion and depolymerization of solid tissues, but may have an adverse effect on the chemical composition of the cells being measured, so it is necessary to combine the purpose of the experiment with the selection of a suitable single-cell suspension preparation method.

Precautions:
1. fresh tissue specimens should be processed and preserved in time, so as not to leave the tissue at room temperature for too long, producing central tissue necrosis or cellular self-melting, which will affect the results of FCM assay;
2. the enzymatic method should pay attention to the selection of conditions and influencing factors, as well as pay attention to the effect of enzyme solvent, digestion time, pH, concentration and other methods on the enzymatic digestion method;
3. different preparation methods should be selected for different solid tissues, such as cell-rich tissues ---- lymphosarcoma, optic neuroblastoma, brain tumors, undifferentiated tumors, medullary tumors, and some soft-tissue sarcomas, etc., where a large number of high-quality, monodisperse cells can be obtained by simple mechanical methods;
4. When using enzymatic methods, attention should be paid to the selection of enzymes, such as tumors containing a large amount of connective tissue ---- esophageal cancer, breast cancer, skin cancer, etc., should be selected for collagenase digestion.

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