Sorting Mouse CD4+ CD25+ Inhibitory Cells by AUTOMACS System Automatic Sorting System

Sorting mouse CD4+ CD25+ inhibitory cells by PriCells-AUTOMACS automatic sorting system
The negative cell (CD4 + CD25 - ) obtained by this method can be used as a reaction cell or a control cell.
Experimental Materials:
Mouse
2. HBSS Wash Buffer
3. MACS buffer
4. CD8a (Ly-2) magnetic beads
5. Binding buffer
6. biotin-anti-CD25 (7D4)
7. Streptavidin-PE
8. Anti-PE beads
9. Complete RPMI medium
10. Mouse CD3 + T cell enrichment column and 1 × purification column buffer
11. 15ml centrifuge tube, sterile
12. autoMACS system and sorting column
13. 30mm nylon mesh or 40mm pre-separation filter
experimental method:
1. Prepare a single cell suspension with lymph nodes from 20 mice and remove red blood cells.
2. The cells were suspended in 20 ml of HBSS wash buffer and counted in a hemocytometer.
3. Centrifuge the cell suspension at 200 °C for 10 min at 4 °C. During the centrifugation, three mouse CD3 + T cell purification columns were prepared and washed 3 times with 1 x purification column buffer as directed by the instructions. Discard the eluent and place a 15 ml centrifuge tube under each column.
4. Suspend the cells with 3 ml of 1X purification column buffer and 3 ml of HBSS wash buffer. After passing the cells through a 30 mm nylon mesh or a 40 mm pre-separation filter, 2 ml of cells were added to each purification column. Incubate for 15 min at room temperature.
5. Wash the purification column 4 or 5 times with 2 ml of 1X purification column buffer to elute T cells. After counting with a hemocytometer, the cell suspension was centrifuged at 200 g for 10 min at 4 °C.
6. The pellet was suspended at a ratio of 10 8 cells plus 0.9 ml MACS buffer. 0.1 ml of CD8a (Ly-2) magnetic beads were added per 10 8 cells and incubated at 4 ° C for 15 min.
7. Centrifuge at 200g for 10min at 4°C.
8. Suspend the cells in MACS buffer to a cell concentration of 10 8 cells/ml. The cells were passed through a 30 mm nylon mesh or a 40 mm pre-separation filter. Place the cells in the loading channel of autoMACS. Select the depletes program (CD4 + T cells will elute from the negative channel.
9. The cell count was recovered and centrifuged at 200 g for 10 min at 4 °C.
10. Suspend the cells in binding buffer to a concentration of 10 8 cells/ml. 15 mg of biotin-anti-CD25 (7D4) was added per 10 8 cells. Incubate at 4 ° C for 15 min. Centrifuge at 200 g for 10 min at 4 °C.
11. Suspend the cells in binding buffer to a concentration of 10 8 cells/ml. 7.5 mg of Streptavidin-PE was added per 10 8 cells. Incubate at 4 ° C for 15 min.
12. Centrifuge at 200g for 10min at 4°C.
13. Suspend the cells in MACS buffer to a concentration of 10 8 cells/ml. 0.1 ml of anti-PE magnetic beads were added per 10 8 cells. Incubate at 4 ° C for 15 min.
14. Centrifuge at 200g for 10min at 4°C.
15. Suspend the cells in MACS buffer to a concentration of 10 8 cells/ml. Place the cells in the loading channel of autoMACS. The potseld program was selected (CD4 + CD25 + cells will elute from the positive channel and CD4 + CD25 - cells will elute from the negative channel.

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