Basic plan
Mononuclear cell isolation and culture were prepared from mouse spleen, thymus and lymph nodes.
Experimental Materials
1. RPMI-5 or DMEM-5 complete medium.
2. Freshly isolated mouse organs: ≤ 6 week old mouse thymus; 6 weeks to 6 months old mouse spleen and lymph nodes.
3.60mm × 15mm culture dish.
4. Scissors and tweezers (stored in a 70% ethanol beaker).
5. A 6 ml syringe with a 19G needle.
6.200 μm nylon filter.
experimental method
1. Freshly separated organs were placed in 60 mm x 15 mm petri dishes (one dish per organ) containing 3 ml of RPMI-5 or DMEM-5 complete medium, and the organs were cut with scissors.
2. Using a 6 ml syringe plunger, squeeze the tissue block against the bottom of the dish until only the fibrous tissue remains.
3. The tissue suspension was repeatedly aspirated several times with a 6 ml syringe containing a 19G needle to further pulverize the tissue mass.
4. The cell suspension was filtered through a 200 μm nylon mesh into a centrifuge tube. Wash the dish once with about 4 ml of complete medium and repeat as needed, then add the wash solution through a strainer to the centrifuge tube.
5. Centrifuge at 1000 r/min (200 g) for 10 min in a Sorvall H-1000B centrifuge and discard the supernatant (if red blood cells and dead cells need to be removed, see Supplementary Protocol). The pellet was resuspended in 20 ml of complete medium, centrifuged, and resuspended in an appropriate amount of medium for counting.
6. If the cells are not treated immediately, the best way to preserve cell viability is to place the cell suspension in ice. This treatment also reduces cell loss caused by cell adhesion.
appendix:
RPMI-5 complete medium:
RPMI1640 medium (Life Technologies)
5% FBS, heat inactivated at 56 ° C for 1 h
10mmol/L HEPES
20μg/ml gentamicin sulfate
50μmol/L 2-ME
Filtration sterilization
PriCells offers products
1. Various primary cell special basal media;
2. Various primary cell culture special additives;
3. Primary cell isolation kit;
4. Primary cell identification kit;
5. Primary cell transfection kit;
6. Primary cell total protein extract;
7. Primary cell total RNA;
8. Primary cell total DNA;
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