The streaming experiment is about to begin. Are you ready to analyze the sample?

First, the preparation of the cells

Test 1: Cell preparation for tissue culture cells

material

Complete medium flow cytometry staining solution 15 or 50 ml sharp bottom centrifuge tube

experiment procedure

1. For suspension cultured cells, the cells are dispensed into a sharp-bottomed centrifuge tube, the cells are counted and analyzed for activity, and then proceeds to step 3;

2. For adherent cultured cells, use a complete medium to blow up and blow up cell aggregates from the culture dish, then dispense the cells into a sharp-bottomed centrifuge tube, count the cells and analyze the activity, and proceed to step 3;

3. The cells were centrifuged and then resuspended in a flow cytometric staining solution to a final concentration of 2 x 107 cells/ml.

Test 2: Cell preparation for lymphoid tissue

material

60×15 mm tissue culture dish

3 ml syringe

Cell strainer (blood nylon strainer)

Flow cytometry staining solution

15 or 50ml tipped centrifuge tube

experiment procedure

1. Tissues were collected (collected tissues (spleen, lymph nodes, thymus) were placed in a cell culture dish and squeezed with a 3 ml syringe plunger to make a single cell suspension using 10 ml of staining buffer;

2. The cells were collected by adding 10 ml of staining solution, and the cell suspension was passed through a nylon mesh to remove agglomerated cells and cell debris, and the cell suspension was collected in a pointed bottom centrifuge tube;

3. Centrifuge the cell suspension at 4 ° C for 4-5 minutes (300-400 g), discard the supernatant;

4. Resuspend cell pellet, cell count and activity assay;

5. The cells were centrifuged according to step 3 and then resuspended in a suitable volume of staining solution to a final concentration of 2 x 107 cells/ml.

Trial 3: Cell preparation of non-lymphoid tissues

material

Scissors or surgical blades

PBS

60×15 mm tissue culture dish

3 ml syringe

Cell strainer (blood nylon strainer)

Flow cytometry staining solution

15 or 50ml tipped centrifuge tube

experiment procedure

1. Collect tissue and cut into small pieces of 2-4 mm with scissors or a scalpel;

2. According to the instruction manual of the enzyme, add an appropriate amount of the enzyme diluted with PBS and incubate.

3. The cells were gently blown off with a pipette and filtered to remove agglomerated cells and cell debris;

4. Centrifuge the cell suspension at 4 ° C for 4-5 minutes (300-400 g), discard the supernatant;

5. Resuspend the cell pellet with PBS to remove the remaining enzyme solution;

6. Repeat step 4;

7. Repeat steps 5 and 6;

8. Resuspend the cell pellet with flow staining, cell count and activity analysis;

9. The cells were centrifuged according to step 4 and then resuspended in a suitable volume of staining solution to a final concentration of 2 x 107 cells/ml.

2. It is best to remove red blood cells before analyzing the lymphatic tissue cell suspension on a flow cytometer.

Test 1: Lysis of red blood cells in mouse spleen cells:

material

1×PBS

eBioscience red blood cell lysate

50ml tipped tube

1. The spleens of the mice were collected and made into a single cell suspension;

2. The cells were pelleted by centrifugation at 4 ° C (300-400 g), and the supernatant was discarded;

3. Resuspend the cells with 5 ml of spleen red blood cell lysate;

4. Incubate for 4-5 minutes at room temperature (also on ice);

5. Stop the reaction by adding 20-30 ml PBS;

6. The cells were pelleted by centrifugation at 4 ° C (300-400 g), and then the cell pellet was resuspended in a suitable volume of buffer to continue the next experiment;

7. cell counts.

Test 2: Red blood cell lysis in mouse blood

material

1×PBS

eBioscience Erythrocyte Lysate (Cat.No.00-4333)

50ml tipped tube

1. 10 ml of red blood cell lysate was added to the blood of 1 ml of mice;

2. Incubate for 4-5 minutes at room temperature (also on ice);

3. Stop the reaction by adding 20-30 ml PBS;

4. The cells were pelleted by centrifugation at 4 ° C (300-400 g), and then the cell pellet was resuspended in a suitable volume of buffer to continue the next experiment;

5. cell counts.

Trial 3: Lysis of red blood cells in human peripheral blood

material

1×PBS

eBioscience red blood cell lysate

50ml tipped tube

1. 10 ml of red blood cell lysate was added to 1 ml of human blood;

2. Incubate for 10 minutes at room temperature (do not exceed 15 minutes);

3. Stop the reaction by adding 20-30 ml PBS;

4. The cells were pelleted by centrifugation at 4 ° C (300-400 g).

The cell pellet is then resuspended in a suitable volume of buffer and the next step is continued;

5. cell counts.