Method for preparing survival single cells based on grinding
A single-cell, grinding device technology, applied in the direction of cell dissociation methods, sterilization methods, biochemical equipment and methods, etc., can solve problems such as instability, complicated operation process, and unstable yield, and achieve a high proportion , high single cell rate and high survival rate
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Embodiment 1
[0041] In this embodiment, the traditional net rubbing method is used to prepare single cell suspension, and the specific operation method steps are as follows:
[0042] In the first step, the nylon mesh is firmly fixed on the opening of the sterile container. The sterile container used in this embodiment is a small beaker, and the nylon mesh adopts a specification of 300 mesh.
[0043] In the second step, the first muscle tissue piece is added to the loosening reagent used in this application and soaked for 20 min. The loosening reagent is composed of neutral protease at a final concentration of 2 mg / mL and type I collagenase at a final concentration of 0.09 mg / mL. , sodium chloride with a final concentration of 10 mg / mL, and magnesium chloride with a final concentration of 5 mM / L.
[0044] In the second step, place the first piece of loosened muscle tissue on a nylon net, rub the muscle tissue repeatedly with a grinding stick or other objects that are convenient for kneading...
Embodiment 2
[0049] In the first step, soak the 3rd muscle tissue piece and loosening reagent for 20min, said loosening reagent consists of neutral protease at a final concentration of 2mg / mL, type I collagenase at a final concentration of 0.09mg / mL, and a final concentration of 10mg / mL. mL of sodium chloride, 5mM / L of magnesium chloride.
[0050] In the second step, filter out the muscle tissue, transfer it to a grinder, and rotate the grinding rod until the tissue is homogenized visually; keep the muscle tissue moist during grinding, and add an appropriate amount of loosening reagent in time to continue grinding until it is homogenized .
[0051] In the third step, add enough normal saline to rinse the grinder, and filter the cell suspension obtained after washing through a 200-mesh nylon mesh;
[0052] In the fourth step, the filtered cells are centrifuged at 1600 rpm for 5 min;
[0053] The fifth step is to remove the supernatant, wash it with PBS, and resuspend it with PBS for later...
Embodiment 3
[0056] This example is a method for preparing surviving single cells using a grinding device independently designed by the applicant. Figure 1-Figure 6 As shown in the grinding device shown, the method is specifically: a method for preparing surviving single cells based on grinding, which specifically includes the following steps:
[0057] Step ST100, preparing grinding materials, including sterilized living biological tissue pieces, loosening reagent and grinding device; the loosening reagent consists of neutral protease at a final concentration of 2 mg / mL, type I collagenase at a final concentration of 0.09 mg / mL, Sodium chloride with a final concentration of 10mg / mL and magnesium chloride with a final concentration of 5mM / L. It is worth noting that the corresponding tissue in this example is muscle tissue, and the concentration of the relevant enzymes is measured according to the maximum concentration. If the tissue to be processed is an easier part, such as liver, spleen,...
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Abstract
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