The basic techniques for preparing cell suspensions from mouse lymphoid organs are first described, including the removal of erythrocytes and dead cells
The method for removing erythrocytes and dead cells is described first.
Author: J.E. Collier et al, Translator: Xuitao Cao et al. This experiment is from the "Compact Immunology Laboratory Guide".
Operation method
Isolation experiments of single nucleated cells in mice Move Basic program for the preparation of cell suspensions from the spleen, thymus and lymph nodes. Materials RPMI-5 or DMEM-5 complete culture medium Freshly isolated mouse organs: mouse thymus < 6 weeks of age; mouse spleen and lymph nodes from 6 weeks to 6 months of age 60 mmX 15 mm Petri dishes Scissors and valvator (stored in a beaker with 70% ethanol) 6 ml syringe with 19 G needle 200um nylon filter 1 . Place the freshly isolated organs into 60 mm X 15 mm Petri dishes (one dish for each organ) containing 3 ml of RPMI-5 or DMEM-5 complete medium and cut the organs with scissors. 2 . Using the plunger of a 6 ml syringe, squeeze the tissue mass firmly toward the bottom of the petri dish until only fibrous tissue remains. 3 . The tissue suspension is repeatedly aspirated several times with a 6 ml syringe fitted with a 19 G needle to further crush the tissue mass. 4 . Strain the cell suspension through a 200 Mm nylon strainer into a centrifuge tube. Wash the dish once with approximately 4 ml of complete medium, repeat if necessary, and add the washings to the centrifuge tube through the strainer. 5. Centrifuge in a Sorvall H-1000B centrifuge at lOOOr/min (200 g) for IOmin and discard the supernatant (if necessary to remove erythrocytes and dead cells, see auxiliary protocol). The precipitate is resuspended in 20 ml complete medium, centrifuged, and resuspended in an appropriate amount of medium for counting (Appendix 3A). 6 . If the cells cannot be processed immediately, the best way to maintain cell viability is to place the cell suspension on ice. This treatment also reduces cell loss due to cell walling (Tip 2. 3). It is advisable to remove red blood cells (RBCs) from spleen cell suspensions before performing a lymphocyte count. Thymus and lymph node cell suspensions do not require RBC removal. Subpopulation isolation of splenocytes also requires removal of RBCs. V A C K lysis buffer 1 . Suspend spleen cells in approximately 5 ml of Lysis Buffer per spleen; a 12 ml tube can be used to lyse one or two spleens, or more spleens can be lysed in a 50 ml centrifuge tube. 2 . Incubate at room temperature for 5 min with shaking. 3 . Add the wash solution until the vessel is full and centrifuge at 200 g for IOmin on a low speed centrifuge and discard the supernatant. Wash the precipitate once more and resuspend with appropriate amount of medium for further operations (e.g. dead cell removal, cell counting, or fine cell counting). High density solution: Ficoll-Paque (Ficoll and pantothenic acid; Pharmacia LKB) or Lympholyte--M (Cedarlane) Note: The densities of Flcoll-Paque and Lympholyte-M are temperature dependent; the high-density solutions used in this method and other commercial products are suitable for use at room temperature. Therefore, care should be taken to keep the cell suspension, centrifugation and high density solution at room temperature. Failure to do so may result in loss of viable cells at the density interface as they sink to the bottom of the centrifuge tube. For more product details, please visit Aladdin Scientific website.
(e.g., dead cell removal, cell counting, or cell separation). The basic principle of the method described below is based on the difference in density between live (less dense) and dead (more dense) cells, which facilitates the separation of live lymphocytes from erythrocytes.
12m l or 50m l polypropylene centrifuge tubes (better than polystyrene centrifuge tubes)