Isolation of mouse mononuclear cells

Isolation of mouse mononuclear cells

Basic plan:
Preparation of cell suspension from spleen, thymus and lymph nodes
Experimental Materials:
1. RPMI-5 or DMEM-5 complete medium
2. Freshly isolated mouse organs: mouse thymus ≤ 6 weeks old; mouse spleen and lymph nodes from 6 weeks to 6 months old
3. 60mm×15mm Petri Dish
4. Scissors and tweezers (stored in a 70% ethanol beaker
5. 6ml syringe with 19G needle
6. 200μm nylon filter
Experimental method:
1. Put the freshly separated organs into a 60mm×15mm petri dish (one petri dish for each organ) containing 3ml of RPMI-5 or DMEM-5 complete medium, and cut the organs into small pieces with scissors.
2. Use the plunger of a 6ml syringe to forcefully squeeze the tissue block towards the bottom of the petri dish until only fibrous tissue remains.Petri Dish 60,Petri Dish 60mm,Petri Dish Price,petri dish petri,Petri Dish Sterile
3. Use a 6ml syringe equipped with a 19G needle to repeatedly suck the tissue suspension several times to further crush the tissue clumps.
4. Filter the cell suspension through a 200μm nylon strainer into a centrifuge tube. Wash the petri dish once with about 4ml of complete medium, repeat if necessary, and then add the washing liquid to the centrifuge tube through the strainer.Petri Dish 60,Petri Dish 60mm,Petri Dish Price,petri dish petri,Petri Dish Sterile
5. Centrifuge in a Sorvall H-1000B centrifuge at 1000r/min (200g) for 10 minutes and discard the supernatant (if red blood cells and dead cells need to be removed, see the supplementary protocol). Resuspend the pellet in 20ml complete medium and centrifuge, then resuspend it in appropriate medium for counting.
6. If the cells cannot be processed immediately, the best way to preserve cell viability is to place the cell suspension in ice cubes. This treatment can also reduce cell loss due to cell adhesion.Petri Dish 60,Petri Dish 60mm,Petri Dish Price,petri dish petri,Petri Dish Sterile