Collagenase IV

Protocol for Single Cell Isolation by collagenase IV for Flow Cytometry Analysis ^[1]:

1. Endometrial samples were minced into fragments of 1 mm3 and digested for 30 minutes at 37℃ with 1 mg/mL of collagenase IV, 0.2 mg/mL of DNase, and 1 mg/mL of bovine serum albumin DPBS.
2. The cell suspension was passed through a mesh, washed in PBS, treated with ACK buffer (0.01 M KHCO₃-buffered 0.16 M, NH₄Cl, 0.1 mM ethylenediaminetetraacetic acid [EDTA]) to remove red blood cells, washed and resuspended in PBS.
3. Isolated cells were incubated with human immunoglobulin for 10 minutes and stained with fluorescent antibodies against specific antigens of different immune cell populations.
4. After 30 minutes of incubation at 4℃, the cells were washed, resuspended in fluorescence-activated cell sorter (FACS) buffer, and analyzed on FACS sort flow cytometer. Unlabeled cells and the respective isotype antibodies were used as a control.

This protocol only provides a guideline, and should be modified according to your specific needs.

Protocol for Isolation and extraction of primary mouse hepatocytes by collagenase Ⅳ perfusion ^[2]:

Two step perfusion

(1)   Preparation: The infusion solution and DMEM high-sugar culture were preheated in a water bath at 42℃ for 30 min to prepare the animal experimental table and surgical instruments, and the aseptic operating table was irradiated with ultraviolet light for 30 min. The peristaltic pump tube circulates with collagenase infusion 1 to prevent bubbles.

(2)   Solution preparation: Collagenase infusion 1: EDTA 0.5 mmol/L, D-Hanks solution 10 mL; Collagenase infusion 2: protease E₃ 2mg, Hanks solution 8mL; Collagenase infusion solution 3: Collagenase Ⅳ 4.8 mg, Hanks solution 12.5 mL; Cleaning solution: DNase I 0.02 mg/mL, Grignard equilibrium salt solution 50 mL; Purified solution :50% Silicated polyvinylpyrrolidone (PVP) density gradient centrifugation agent; Complete medium: Insulin 0.5μg /mL, dexamethasone 100nmol /L, cyanstreptomycin 1%, fetal bovine serum 10%, DMEM high-glucose medium 200mL.

(3)   Infusion: The mice were killed by CO₂ asphyxiation and soaked in alcohol for 10~20s.Transfer to sterile operation table. Dissecting mice, exposing the abdominal cavity and the entire chest cavity. Ligate the superior hepatic segment of the inferior vena cava and enter the needle horizontally from the inferior vena cava. Open perfusion and make a small incision in the portal vein. Perfusion velocity 3 mL/min;9 ml of collagenase infusion 1 is consumed, and the outflow liquid is clarified, that is, the infusion is stopped. Transfer the pump tube to collagenase infusion 2 and continue perfusion, consuming approximately 8 (or 15) mL of collagenase infusion 2. Liver gradually swelling, with a glass minute hand gently press the liver, concave and slow rebound, you can. Replace with collagenase infusion 3 and consume 11 mL (or 21 mL). If more cells are needed, the perfusion volume and time can be increased.

(4)   Separation: The aggregation of the fiber bundles connecting each liver lobe was clipped with tweezers, cut along the connective tissue, put into a centrifugal tube containing collagenase infusion solution 3, sealed and pre-heated at 42 ℃ for 5min.The liver was immersed in a petri dish containing 10% fetal bovine serum medium for liver tear (gently peel the liver leaf envelope, then grasp the fiber bundle with tweezers, shake to disperse the remaining cells), and filtered through a 200-mesh filter, the filtrate was liver cell suspension.

(5)   Purification: The filtered liver cell suspension was transferred to a 50mL centrifuge tube and centrifuged at low temperature and speed (4 ℃, 50×g, 3 min). Discard the supernatant, add 15 mL cleaning solution and blow gently for 3 times, repeat centrifugation for 3 times. Discard the supernatant, add 10 mL of complete medium to resuspension cells, and slowly add to 10 mL of purified solution, centrifuge (4℃, 400×g, 10 min), discard the upper layer, the remaining cell precipitate is liver parenchymal cells, add 5 mL of complete medium to resuspension cells.

This protocol only provides a guideline, and should be modified according to your specific needs.

References:

[1].  Gnainsky Y, Granot I, et,al. Local injury of the endometrium induces an inflammatory response that promotes successful implantation. Fertil Steril. 2010 Nov;94(6):2030-6. doi: 10.1016/j.fertnstert.2010.02.022. Epub 2010 Mar 24. PMID: 20338560; PMCID: PMC3025806.

[2].Ren Wenjie, Lin Zhexuan. Trypsin and collagenase perfusion method of separation of the original generation of liver cells in mice to compare [J]. Journal of shantou university medical college, 2022, 35 (4) : 204-209. The DOI: 10.13401 / j.carol carroll nki jsumc. 2022.04.003.