Brief Introduction To The Methods of Mouse Primary Cell Isolation

In modern biomedical research, mice are widely used as model organisms, and the techniques for isolating and culturing their primary cells are of great significance for both basic and applied studies. For newcomers who have just entered the laboratory, mastering the methods for extracting primary cells from mice is an important first step toward scientific research. However, when facing a mouse, many beginners often feel overwhelmed by the question of how to obtain primary cells from its various tissues and organs.

Primary cells refer to cells that are directly isolated from body tissues and cultured in vitro. They retain many characteristics of cells in vivo, making them ideal materials for studying cell biology, pharmacology, gene expression, and other fields. This article briefly outlines the basic methods commonly used in laboratories for isolating primary cells from mouse tissues and organs. It aims to help beginners get started smoothly and quickly with experimental operations, thereby reducing the confusion often experienced during initial laboratory work.

I. Preparation of Mouse Liver Immune Cell Suspension

Liver Harvesting: After anesthetizing the mouse with isoflurane, euthanize via cervical dislocation. Disinfect the mouse by immersing it in 75% ethanol. Dissect the mouse to harvest the liver and remove the gallbladder.

 

Digestion: Transfer the liver to a petri dish and finely mince it using scissors. Resuspend liver fragments in 15 mL of digestion solution (0.5 mg/mL Type IV collagenase and 20 ng/mL DNase I), and transfer to a 50 mL centrifuge tube. Label the tube and lay it flat on a shaker. Digest at 37°C, 250 rpm for 30 minutes.

 

Washing: After digestion, fill the tube with pre-chilled 1× PBS to wash the liver cells. Centrifuge at 1500 rpm, 4°C for 5 minutes. Discard the supernatant.

 

Grinding: Resuspend the pellet in 2 mL of pre-chilled 1× PBS. Filter the cell suspension through a nylon mesh into a new 15 mL centrifuge tube. Residual undigested tissue on the mesh is ground in 5 mL of pre-chilled 1× PBS using the plunger of a 1 mL syringe until fully dispersed. Transfer the liquid to the centrifuge tube and wash the mesh with an additional 5 mL of pre-chilled 1× PBS. Collect all liquid and centrifuge at 1500 rpm, 4°C for 5 minutes.

 

Immune Cell Isolation: Resuspend the pellet in 5 mL of mouse lymphocyte separation solution (equilibrated to room temperature). Slowly layer 1 mL of serum-containing RPMI 1640 medium on top. Perform density gradient centrifugation at 2200 rpm, 25°C for 30 minutes with acceleration/deceleration set to 0. After centrifugation, collect the white immune cell layer between the two phases. Wash cells with pre-chilled 1× PBS, centrifuge at 1500 rpm, 4°C for 5 minutes.

 

Resuspension & Counting: Resuspend in 1 mL of pre-chilled 1× PBS. Dilute 10 μL of suspension into 990 μL of 1× PBS (1:100) and count cells using a hemocytometer. Store at 4°C until use.

 

II. Preparation of Mouse Lung Single-Cell Suspension

Lung Harvesting: Euthanize the mouse after isoflurane anesthesia via cervical dislocation. Disinfect in 75% ethanol, dissect, and remove the lung tissue, trimming off excess tissue.

 

Digestion: Mince the lungs and resuspend fragments in 5 mL of digestion solution (0.5 mg/mL Type IV collagenase and 20 ng/mL DNase I). Transfer to a 15 mL tube, label, and lay flat on a shaker. Digest at 37°C, 250 rpm for 60 minutes.

 

Washing: Fill with pre-chilled 1× PBS, centrifuge at 1500 rpm, 4°C for 5 minutes, and discard the supernatant.

 

Grinding: Resuspend the pellet in 2 mL of pre-chilled 1× PBS and filter through a nylon mesh. Grind remaining tissue in 5 mL of PBS with a syringe plunger and collect as above. Centrifuge at 1500 rpm, 4°C for 5 minutes.

 

RBC Lysis: Add 1 mL ACK lysis buffer to the pellet, mix thoroughly, and incubate at room temperature for 5 minutes.

 

Stop Lysis: Add 9 mL of pre-chilled 1× PBS to stop the reaction, then centrifuge at 1500 rpm, 4°C for 5 minutes.

 

Resuspension & Counting: Resuspend in 1 mL of 1× PBS, dilute, count, and store at 4°C.

 

III. Preparation of Mouse Colon Immune Cells

Colon Harvesting: Euthanize via isoflurane and cervical dislocation, disinfect, and harvest the colon. Open longitudinally, wash thoroughly with 1× PBS until clean.

 

Digestion: Mince the colon into ~1 mm³ pieces and resuspend in 10 mL of digestion buffer (0.1% collagenase I and 3% dispase II). Transfer to 15 mL tubes, label, and digest at 37°C, 250 rpm for 25 minutes.

 

Immune Cell Isolation: Filter digested tissue through a nylon mesh, centrifuge at 1500 rpm, 4°C for 5 minutes. Resuspend in 5 mL lymphocyte separation buffer, then carefully layer 1 mL RPMI 1640 medium on top. Centrifuge at 2200 rpm, 25°C for 30 minutes with no acceleration/deceleration. Collect the immune cell layer, wash, centrifuge again.

 

Resuspension & Counting: Resuspend in 1 mL of 1× PBS, count, and store at 4°C.

 

IV. Preparation of Mouse Spleen (Thymus / Mesenteric Lymph Node) Cell Suspension

Spleen Harvesting: Dissect and clean the spleen from fat and connective tissue post euthanasia.

 

Grinding: Grind spleen tissue in 5 mL of 1× PBS using a syringe plunger in a nylon mesh. Transfer liquid to a centrifuge tube, wash mesh with additional 5 mL PBS. Centrifuge at 1500 rpm, 4°C for 5 minutes.

 

RBC Lysis: Add 1 mL ACK buffer, resuspend thoroughly, incubate 5 minutes at room temperature.

 

Stop Lysis: Add 9 mL of 1× PBS, centrifuge at 1500 rpm, 4°C for 5 minutes.

 

Resuspension & Counting: Resuspend cells in 1 mL PBS, count, store at 4°C.

 

V. Preparation of Mouse Bone Marrow Single-Cell Suspension

Bone Isolation: Harvest femur and tibia post euthanasia. Clean off muscle tissue and place in cold 1× PBS.

 

Bone Marrow Collection: Cut both ends of the bones, flush bone marrow using 1× PBS with a syringe into 15 mL tubes. Repeat until bones turn white. Centrifuge at 1500 rpm, 4°C for 5 minutes.

 

RBC Lysis: Add 1 mL ACK buffer, mix thoroughly, incubate for 8 minutes at room temperature.

 

Stop Lysis & Filtering: Add 9 mL PBS, filter through nylon mesh into a new tube to remove debris. Centrifuge again.

 

Resuspension & Counting: Resuspend in 1 mL PBS, count, store at 4°C.

VI. Preparation of Mouse Bronchoalveolar Lavage Fluid (BALF) Cells

Mouse Fixation: After euthanasia, disinfect and fix the mouse on a foam board with a syringe needle.

 

Tracheal Cannulation: Expose the trachea, insert the outer catheter of a venous indwelling needle, withdraw steel core, and ligate to secure.

 

Lavage: Inject 1 mL pre-chilled PBS via syringe through the tracheal catheter, aspirate back 3–4 times. Collect lavage fluid into 15 mL tubes. Repeat to collect 8–10 mL.

 

Centrifugation: Centrifuge at 1500 rpm, 4°C for 5 minutes.

 

Resuspension & Counting: Resuspend in 1 mL PBS, count, and store at 4°C.