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Storage:Store at RT，avoid light，2 years.
This product is used for the isolation of goose spleen lymphocytes.
This product is susceptible to bacterial infection and should be operated under sterile conditions. Operate under sterile conditions and store at room temperature after opening. If stored at 4℃, the separation liquid is prone to white crystallization, which affects the separation effect.
Kit content:
Sample diluent 200mL
Cell cleaning Solution 200mL
Lymphocyte isolation solution 200mL
Reagent F 200mL
Manual 1 Copy
Preparation of Spleen tissue single-cell suspension
Note:A. The whole process and the reagents required require a sterile environment.B. This kit does not contain collagenase. If single-cell suspension is prepared by enzyme digestion method, please purchase different types of collagenase according to the requirements of each laboratory.
1. Cutting method:
After the tissue blocks were put into the plate, a small amount of liquid F and 20% fetal bovine serum were added; The tissue was cut to homogenate shape with ophthalmic scissors, and 5mL liquid F and 20% fetal bovine serum were added. Absorb the tissue homogenate with a straw and filter it into the test tube with a 100-mesh stainless steel filter (purchased separately); Centrifuge precipitation 1500 RPM ×3 min, and then clean with cell cleaning solution 3 times, each time at 500rpm short-time low-speed centrifuge to remove cell debris, filter with 200 mesh stainless steel filter (purchased separately) to remove cell mass. The cells were counted and the cell concentration was adjusted to (2-5) ×107 cells /mL. Placed at room temperature to measure cell viability. Before separation, 20% fetal bovine serum or sample diluent was used to suspend single-cell suspension with cell concentration of 2×108~1×109 /mL.
2. Homogenizer method:Use ophthalmic scissors to cut the tissue into small pieces; Put into 70mL tissue grinder, add 2mLF liquid and 20% fetal bovine serum; Slowly turn the pestle and grind until homogenized; Rinse the pestle with 5mLF liquid and 20% fetal bovine serum; The cell suspension is collected and filtered through a 200-mesh stainless steel filter (purchased separately). Centrifuge precipitation 800rpm×2min, then washed with cell cleaning solution for 3 times, centrifuge precipitation. The cells were counted and the cell concentration was adjusted to (2-5) ×107 cells /mL. Placed at room temperature to measure cell viability. Before separation, 20% fetal bovine serum or sample diluent was used to suspend single-cell suspension with cell concentration of 2×108~1×109 /mL.
3. Enzyme digestion method:
Dilute the collagenase with liquid F, the final concentration is 400 U/mL, and the ice bath is reserved. An appropriate size petri dish was taken for operation: animal spleen was crushed with tweezers, diluted collagenase was added, and digested at 37℃ for 20 minutes. Filter with 100 or 200 mesh stainless steel filter (purchased separately), centrifuge precipitation 800prm×2min, then wash with cell cleaning solution for 3 times, centrifuge precipitation. The cells were counted and the cell concentration was adjusted to (2-5) ×107 cells /mL. Placed at room temperature to measure cell viability. Before separation, 20% fetal bovine serum or sample diluent was used to suspend single-cell suspension with cell concentration of 2×108~1×109 /mL.
Cell count method:
Cell counting is a method used to count the number of cells in cell suspension. It is generally carried out using a counting plate (blood cell counting plate). It can be used to count the number of cells in the cell suspension prepared before the separation (loose) cell culture inoculation, and can be used to count the number of cells in the culture. A dispersed cell suspension must be prepared regardless of the object being counted.
Counting and calculating process
1) Place a cover slide for counting in the center of the cell counting plate.
2) Draw cells with a glass siphon and let the siphon drain the suspension on the cover glass or at the concave of the counting plate on the lower side until the cover glass is filled with liquid.
3) Count the total number of cells in the square under the microscope. Count only the upper and left lines for cells pressing the line, and count individual cells for cell clusters.
4) Count the density of cell suspension according to the following formula: cell density = (total number of 4 large cells /4) ×104 cells /mL× dilution
The formula is multiplied by 104 because the volume of each large cell in the counting plate is:
1.0mm (L) ×1.0mm (W) ×0.1mm (H) = 0.1mm3 and 1mL = 1000mm3
Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.
