Guidelines for the preparation of spleen single-cell suspensions (enzymatic digestion)

The mouse spleen is an important immune organ located in the left abdominal cavity of the mouse, near the left side of the stomach, in an oblong shape. The size will vary depending on the breed and health of the mouse, but in general, the spleen length of an adult mouse is approximately between 1.5 cm and 2.5 cm. The color and texture of the spleen may vary depending on the breed and health of the mouse, and it usually has a leaf-like shape and is dark red in color. In experimental research, the isolation, culture, and enumeration of mouse spleen cells is an important method for immunological research, and it is also a prerequisite for flow cytometry analysis.

 

 

1. Consumables and reagents

Table 1 Reference table of some reagent consumables for dissociation of mouse spleen

material	reagent	equipment
15/50 mL centrifuge tubes	1x PBS buffer	Horizontal thermostatic shaker
10 cm Petri dish	RPMI 1640 medium	Hemocytometer
Ophthalmic surgical scissors, eye tweezers	75% alcohol	Electronic pipettes, 1mL tips
100 μm cell sieve	FBS	High-speed centrifuge
10 mL pipette	HBSS equilibrium solution	Vortex instrument
5 mL flow cytometry tubes	Absin Tissue Dissociation Kit	4 °C freezer
	Cell staining buffer	Flow cytometry

 

Table 2 Product information

Catalog number	Product name	specification	use
abs50097	Mouse spleen tissue dissociation kit	10T/25T	Dissociate the tissue
abs7303	5 mL flow cytometry tubes (sterile, enzyme-free, with caps)	500pcs/carton	Flow cytometry tubes
abs9484	RPMI1640 medium	500mL	Preserve cell viability
abs7232	70um cell sieve	100pcs/carton	Supplies
abs7102	15 mL centrifuge tubes (culet-bottom)	100pcs/bag	Supplies
abs7100	50 mL centrifuge tubes (culet-bottom)	40pcs/bag	Supplies
abs962	PBS buffer (1x)	500mL	reagent
abs9475	Cell staining buffer	500mL	reagent
abs9101	Red blood cell lysate (1x)	100mL/500mL	Split red liquor

 

2. Experimental procedures

Preparation before the experiment: preheat the spleen mouse spleen tissue dissociation kit in a 37 degree water bath (after the spleen tissue dissociation reagent is received, it is recommended to divide and cryostore), if the red blood cell lysate is 10 ×, it needs to be diluted into 1 × with ultrapure water in advance. PBS pre-cooled in advance.

1. Dislocate the neck of the mouse to death, disinfect the surface of the mouse with 75% alcohol, as for the tray, the left abdomen is facing up, the tweezers assist the operation, lift the skin in the middle of the left abdomen, cut the skin, the dark red is visible to the naked eye, the long strip-like spleen, cut the peritoneum, lift the spleen with forceps, see the separation, remove the connective tissue and fat as much as possible, rinse with PBS, take about 100mg-200mg of fresh mouse spleen tissue (a 20g mouse, the spleen weight may be about 100mg, The specific situation shall prevail) add to a 50mL sterile centrifuge tube, and thoroughly chop the tissue to 1-2mm with ophthalmic scissors³, add 1mL of mouse spleen tissue dissociation reagent;

2. Place the centrifuge tube on a constant temperature shaker, incubate at 37°C/180rpm for about 30min, digest the spleen tissue of mice, and adjust the digestion time appropriately according to the state of the tissue (the spleen is relatively easy to digest).

 

In the red circle: dead cells In the green circle: live cells

3. After the incubation, the tissue homogenate in the centrifuge tube is filtered with a 70um sterile filter, then the cell culture medium is added to flush the sterile filter, and the filtrate is collected with a 50mL centrifuge tube;

4. Centrifuge the collected filtrate at 1200rpm for 5min and discard the supernatant;

5. Red blood cell lysate can be used to remove red blood cells in the process of tissue dissociation, the specific operation is to add 3mL of 1× red blood cell lysate to the precipitate obtained in step 4, resuspend the precipitate, vortex to mix, incubate at room temperature in the dark for 3-5min, add 2mL of pre-cooled PBS, centrifuge at 1200rpm for 5min, and discard the supernatant. Repeat the wash once;

6. Discard the supernatant, resuspend the cell pellet in cell staining buffer, and adjust the cell concentration to 5-10×10⁶cells/mL, dispense the cell suspension of 100 uL/tube into a 12×75 mm flow cytometry tube for follow-up experiments.

3. Problems and strategies

1. Low cell yield

In general, adult C57BL/6 mice can dissociate about 10×10 spleen⁸–1.4×10⁸cells, if the cell yield is found to be particularly low, it is necessary to pay attention to the complete removal of the fat around the tissue as much as possible during the sample acquisition stage, and to cut the tissue as much as possible to improve the cell yield. In the washing step involved, use a pipette to aspirate the upper layer of fat before decanting the supernatant (trim the tip of the tip to avoid clogging when aspirating)

2. The cell viability is low, and there are many dead cells/debris

First of all, the incubation time of the dissociation reagent should not be too long, long-term incubation will cause the reduction of cell viability and the loss of surface markers, too many dead cells, the release of DNA filaments entangled, resulting in viscous solution, secondly, do not shorten the cleaning steps, cleaning can help remove adhesions, debris and dead cells, the incubation time of red blood cell lysate can not be too long, generally 5min, if the cell viability is still very low, it is recommended to check whether the red blood cell lysate is fresh and effective.

3. Incomplete lysis of red blood cells

Manifests as a noticeable red precipitate after the washing step. Make sure to prepare fresh erythrocyte lysis buffer to lyse at room temperature. Before lysis begins, make sure that the cells are completely resuspended and that the lysis time is not too short.

Fourth, the dissociation effect

In this kit, mouse Spleen tissue was processed into a single-cell suspension and then flow-byme: cell viability, CD45, CD3, CD4, CD8 staining results

Note: This product is only used for scientific research experiments and does not support clinical research

5. Other product recommendations

Catalog number	Product name	specification
abs9907	Flow cytometry absolute counting tubes	50T
abs9477	Mouse Fc receptor blocker	200T/500T
abs9476	Human Fc receptor blocker	50T/200T
abs9488	Sheath fluid	20L
abs9107	Phoboester	1mg/5mg/25mg
abs9108	Ionomycin calcium salt	1mg/5mg
abs970	D-PBS buffer (1 ×, no calcium magnesium)	500mL
abs810012	Brefeldin A	10mg/25mg/50mg
abs812975	Monensin sodium salt	25mg/100mg
abs9475	Cell staining buffer	500mL
abs9110	fixer	50mL
abs9111	Permeabilization agent	150mL
abs7303	5 mL flow cytometry tubes (sterile, enzyme-free, with caps)	1 box
abs1840002	APC Mouse anti-Human CD3 Antibody(3JI3-9)	25T/100T
abs182427	PE Mouse anti-Human CD4 Antibody(5D4)	25T/100T
abs182430	FITC Mouse anti-Human CD8 Antibody(3C4)	25T/100T
abs180046	PE-Cy7 Mouse anti-Human CD25 Antibody(BC96)	25T/100T
abs1840696	FITC Mouse anti-Human CD80 Antibody(2D10.4)	25T/100T
abs182338	APC Rat anti-Mouse CD25 Antibody(4APC61.5)	25T/100T
More......

 

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