Section: 802, (Thursday)

Group: 4

Names: John Gibson, Kyle LaPlant

Instructor: S. Royan, PhD

Date: Sep 19, 2024

University Of Massachusetts,

Lowell, Fall 2024

Immunology Lab Report 1: Mouse Immune System And Spleen Cell Count

Contributions: 

Objective 1st paragraph by Gibson; 2nd paragraph by Kyle LaPlant

Material/Method 1st paragraph by Gibson; 2nd paragraph by Kyle LaPlant

Result 1st paragraph by Gibson; 2nd paragraph by Kyle LaPlant

Discussion 1st paragraph by Gibson; 2nd paragraph by Kyle LaPlant

Picture editing by Gibson

 

Objective

This experiment looked to identify and examine a mouse's major immune organs and tissues, observing the anatomy of the spleen, thymus, and Peyer’s patches. Additionally, lymphocytes were isolated from the dissected spleen and an estimation of total cell count and cell viability was conducted.

Material And Method

An euthanized CD-1 breeder mouse was obtained. Using an ear punch, the mouse was marked as the number 50 mouse with a wedge at the middle left ear lobe edge. A syringe with a needle and 0.5 mL of serum-free DMEM was used to inject the right peritoneal cavity with fluid. Three incisions of the skin from the opening of the urethra were made toward the two knees and the chin while using forceps to pull the skin in the opposite direction of scissors. The skin flaps were pinned to the styrofoam plate with brown paper. The epigastric lymph node was observed and photographed. The abdominal membrane was opened, and the intestines were pushed to the right side of the mouse to expose the spleen attached to the left side of the stomach. The mesenteric lymph nodes on the right side of the mouse were presented and photographed. A pair of scissors cut the base artery of the spleen to free it.

The thymus was located after cutting open the rib cage along the sternum. The left side epigastric (inguinal) lymph node was located underneath the abdominal skin at the bifurcation point. The gastrointestinal tract was excised from the esophagus to the rectum. Peyer’s patches were located and photographed.

The spleen was ground gently with a pair of matt-surfaced glass slides on a petri dish, rinsed with 8 mL of DMEM solution, and stored with the rinsing 8 mL DMEM in a centrifuge tube. The solution was set on ice for 2 minutes to allow connective tissues to settle. After discarding the settled connective tissue, the cellular suspension was centrifuged in 400 g-force to obtain the spleen cells in a pellet. The clear liquid supernatant was discarded, and spleen cells were resuspended in fresh 10 mL DMEM and held on ice. 

100 μL of the cellular suspension of the spleen was combined with 100 μL of Trypan Blue dye, forming a 1:2 dilution in a well plate well. A 10 μL dyed suspension was injected into a hemacytometer chamber with four 0.1μL volume squares. Microscope views of each square, 100x magnification, were photographed with a smartphone. Each square’s viable (transparent bubbles without dye penetration) and dead cells (blue splots of dye penetration and burst) were counted manually on the photographs. This procedure was repeated by diluting another 100 μL cell suspension volume with Trypan Blue dye, the same dilution factor as before, for a second hemacytometer chamber. The two chambers provided a combined eight squares of 0.1μL cellular suspension volumes for counting cells.

Data

Figure 1 shows a photograph of the inguinal (epigastric) lymph node (transparent). Figure 2 showed a photograph of the spleen after a pair of scissors cut the base artery of the spleen and before removing the spleen.

The mesenteric lymph nodes were shown in Figure 3. The thymus was shown in Figure 4 after cutting open the rib cage along the sternum. 

Multiple Peyer’s patches were photographed and shown in Figure 5. Stomach, jejunum, ileum, cecum, colon, and rectum are included in Figure 5. A select microscopy photograph of the second hemacytometer chamber’s 4th square was shown in Figure 6.

As shown in Table A, the cell counts of chamber 1’s squares average 925; the cell counts of chamber 2’s squares average 1145. When averaging chamber 1 and chamber 2’s cell counts, there were, on average, 890 viable cells and 145 dead cells, for a total of 1035 cells per 0.1 μL volume square.

 

The average per-square viable cell count is calculated as the following,

Viable cells: (993 cells + 776 cells + 720 cells + 591 cells) / 4 = 770 cells per square, chamber 1.

Dead cells: (141 cells + 143 cells + 126 cells + 210 cells) / 4 = 155 cells per square, chamber 1.

Viable:(880 cells + 1055 cells + 1087 cells + 1018 cells)/4=1010 cells per square, chamber 2.

Dead cells: (98 cells + 125 cells + 178 cells + 138 cells) / 4 = 135 cells per square, chamber 2.

Average cell counts, combining chamber 1 and chamber 2,

viable cells: (770 cells + 150 cells) = 925 cells; dead cells: (1010 cells + 135 cells) = 1145 cells.

Average of the 2 chamber’s viable and dead cells per square,

viable cells: (770 cells + 1010 cells) / 2 = 890 cells; 

dead cells: (155 cells + 1145 cells) / 2 = 145 cells.

Average of the 2 chambers’ combined cell counts per square,

(925 cells + 1145 cells) / 2 = 1035 cells average per square.

Table A. Cell Count Of Each Hemacytometer Square

Legend: Each 0.1 μL volume square of dyed suspension is counted for viable cells (recorded in red ink) and dead cells (recorded in black ink).  The viable cell counts ranged from 591 to 1087 in a 0.1 μL volume square. The dead cell counts range from 98 to 210 in a 0.1 μL volume square.

Result

As shown in Figure 2, the ear punch identified the mouse as having the number 50 throughout the experiment. The peritoneal injection was practiced, but there was no visible mark of the puncturing of the peritoneal membrane in Figure 1 when the abdominal skin was pulled back and pinned to the side. The mouse’s abdominal and thoracic cavities were dissected and photographed in Figure 1 through Figure 5. The organs and tissues observed were the inguinal lymph node (Figure 1), spleen (Figure 2), the mesenteric lymph nodes (Figure 3), the thymus, the heart, the liver (Figure 4), the stomach, the small intestines, the jejunum, the ileum, the Peyer’s patches, cecum, colon, the rectum (Figure 5). The inguinal lymph node was transparent and not visible to the naked eye in Figure 1 in this particular specimen. Also, in Figure 5, it is discernible that the Peper’s patches occur more frequently in the jejunum than in the ileum section.

The hemacytometer cell counting of the spleen was performed without error incidence. As shown in Table B, the average cell count, viable and dead, of chamber 1, taken from Table A, was 925; the average cell count, viable and dead, of chamber 2, taken from Table B, was 1145. The average cell count in diluted 0.1μL volume is 

(925 cells + 1145 cells) / 2 = 1035 cells.

The dilution factor is 2, so the original cell suspension’s concentration’s cell count per 0.1μL volume is 

1035 cells/0.1μL  x 2 = 2070 cells/0.1μL.

Converting the concentration from 0.1 μL denominator to mL denominator,

2070 cells/0.1 μL x 1000 μL/mL = 2070/0.1 x 1000 cells/mL = 2.07x107 cells/mL

Since all the cells in the spleen were contained in the 10 mL suspension, the total cell count is

2.07x107 cells/mL x 10 mL = 2.07x108 cells.

The cell viability is calculated from Table A’s averaging chamber 1 and chamber 2’s cell counts, on average, 890 viable cells and 145 dead cells, for 1035 cells per 0.1 μL volume square. And viability percentage is 

890 viable cells / 1035 cells x 100 % = 85.99 %.

Table B. Total Spleen Cell Count And Cell Viability

Legend: The A column shows the total number of cells in the diluted sample(0.1 ul) and column B shows total cells of the original sample(0.1 ul) accounting for dilution. Column C is the number of cells/mL and column D shows the total number of cells in the isolated spleen. For columns A-D there are values for Ch.1, Ch.2, and the average for both chambers. Additionally, column E shows the average calculated cell viability from both chambers.

Discussion

Ear punching experimental animals is a technique used by researchers to identify and distinguish animals from each other within a study. The largest benefit of ear punching is that it is a permanent marking that limits human error, such as misplacing animals in the wrong group. This ability to consistently distinguish animals from each other allows researchers to assure that each animal is being exposed to the correct variables or conditions, which adds credibility and accuracy to the overall results.

IP(interperitoneal) injections are another tool commonly used in animal based immunological studies. They are predominantly used because they are minimally invasive and provide a consistent mode of administering substances of interest ranging from antibodies to pharmaceutical drugs (1). Additionally choosing a uniform method of administration limits the number of variability between the conditions of each animal and better isolates the variables of interest. Improper IP injections can cause detrimental damage to the animal or alter the conditions the animal is experiencing, both of which could impact the experimental outcome.

Mice have two primary lymphoid organs, bone marrow and the thymus, which produce lymphocytes of the adaptive immune system. Bone marrow is responsible for the production of both B cells and T cells, with B cells also undergoing maturation. The thymus is then responsible for maturing the unmatured T cells from the bone marrow. In addition to the primary lymphoid organs, mice also possess secondary lymphoid organs which provide place for lymphocyte and antigen interactions. Some of the key secondary lymphoid organs within mice are the spleen, peyer's patches, and lymph nodes such as mesenteric and axillary. The spleen is primarily responsible for the filtration of red blood cells and fighting blood borne pathogens. Peyer's patches play an important role in the GALT, which protects the body from intestinal pathogens (2). Lymph nodes serve as an important junction between the circulatory and lymphatic system.

The inguinal lymph node was not prominently presented in Figure 1 in this particular specimen, likely due to negligible pathogen infection of the mouse in the facility. This is in line with the consensus of the literature (3). Figure 5 shows that the white Peyer’s patches are more prominent and numerous in the jejunum than in the ileum section of the small intestine. This is in line with the consensus of the literature (3).

The total cell count for the spleen was calculated at 2.07x108, which was higher than the consensus literature value (3). However, the value was within reason of explanation, with factors such as mouse size or subjective counting error being possible causes for the increase. As for cell viability, 85.99% is high, likely due to the minimal delay between preparing the cells with Trypan Blue dye and microscopy photography cell counting. If the delay between preparing the cells with Trypan Blue dye and cell counting is prolonged, more cells likely would have died of the toxicity of Trypan Blue and lowered the viability percentage.

Conclusion

In conclusion, this experiment was a total success. The total cell count for the spleen of 2.07x108 is in line with the consensus literature value indicating a healthy mouse with more than a healthy organ size (3). The facility has no pathogenic infections on the mouse, as indicated by the small sizes of secondary lymph nodes on the inguinal and other MALT’s, specifically the Peyer’s patches. The high cell viability, 85.99%, indicated a proficient hemacytometer technique utilization for minimal delay between preparing the cells with Trypan Blue dye and microscopy photography cell counting.

References 

1. Al Shoyaib A, Archie SR, Karamyan VT. Intraperitoneal Route of Drug Administration: Should it Be Used in Experimental Animal Studies? Pharm Res. 2019 Dec 23;37(1):12. doi: 10.1007/s11095-019-2745-x. PMID: 31873819; PMCID: PMC7412579.

2. Reboldi A, Cyster JG. Peyer's patches: organizing B-cell responses at the intestinal frontier. Immunol Rev. 2016 May;271(1):230-45. doi: 10.1111/imr.12400. PMID: 27088918; PMCID: PMC4835804

3. Royan, S.V. & A. Alton. (2024). Immunology Laboratory Manual Fall 2024. [Unpublished Immunology Laboratory Manual]. Department of Biological Sciences, University of Massachusetts, Lowell.