Section: 802, (Thursday)
Group: 4
Names: John Gibson, Kyle LaPlant
Instructor: S. Royan, PhD
Date: Nov 21, 2024
University Of Massachusetts,
Lowell, Fall 2024
Immunology Lab Report 6: Cytotoxicity Assay on Lymphocytes Isolated from Mouse
Contributions:
Objective LaPlant
Material/Method LaPlant
Data/Result Gibson; Revision LaPlant
Discussion a) e) LaPlant; b) c) d) Gibson
Reference LaPlant & Gibson
Objective
The experiment aimed to isolate lymphocytes from the thymus and spleen of a dissected mouse and perform a cytotoxicity assay using Cedarlane’s anti-mouse CD-90.2 (Thy 1.2) monoclonal antibodies alongside complement component proteins. Performing the assay and gathering cell viability via hemocytometer allowed for the evaluation of cytotoxicity of Thy 1.2 and quantification of the number of T-cells in the thymus and spleen.
Material And Methods (6)
Isolation of Lymphocytes from Mouse Spleen and Thymus
A euthanized CD1 strain mouse was placed in a sterile petri dish and soaked with 70% ETOH. Forceps were then used to pinch the skin towards the left side of the abdomen before a small incision was made over the spleen. This was repeated for the neck region, where an incision was made above the thymus. Using fine forceps and scissors, the spleen was then further exposed by pinching and creating an incision in the abdominal wall, which was then used to remove the tissue. The isolated spleen was then trimmed from any unwanted fatty or connective tissue. The thymus was then removed through the previously made incision and also trimmed of unwanted tissue. Both the spleen and thymus were placed in separate sterile Petri dishes with 5 mL of serum-free DMEM.
Two pairs of clean ground glass slides were used to press and grind the organs separately, with the serum-free DMEM in the dish and a pipette being used to rinse off the remaining tissue. The resulting solution of serum-free DMEM and ground organs were then placed into 2 separate 15 mL screw cap centrifuge tubes and mixed by pipetting repeatedly. After letting the solution settle on ice for 2-3 minutes, the supernatant was removed and placed into a new sterile 15 mL tube, with DMEM being used to bring the volume up to 5 mL.
The spleen and thymus cells tubes were then centrifuged for 5 minutes at 400 g (1000-1500 rpm), with the supernatant being discarded after with a 5 mL pipette. The remaining (cell) pellets were resuspended in Cedarlane media without complement, with 10 mL used for the spleen and 5 mL for the thymus.
Cytotoxicity Assay
Four 1.5 mL microfuge tubes were labeled 1-4, with tubes 1 & 2 receiving 0.2 mL of the thymus cell suspension and tubes 3 & 4 receiving 0.2 mL of spleen cell suspension. The tubes were then centrifuged at 4000 rpm for 5 minutes and the supernatant was discarded. The pellets in tubes 1 & 3 were resuspended with 0.2 mL of diluted serum without antibodies as an experimental control, while tubes 2 & 4 were resuspended with 0.2 mL diluted antiserum with anti-Thy antibody. The resulting samples were incubated at 4° C for 30 minutes for antibody bindings. After incubation, they were centrifuged using the same parameters as before, and the supernatant was discarded. The pellets were then resuspended using 0.2 mL of cytotoxicity medium that contained complement components 1 through 9 (C1-C9) proteins and incubated at 37o C for 30 minutes. The tubes were once again centrifuged with the same parameters, and the supernatant was discarded. The pellets were then each resuspended in 0.1 mL of Cedarlane cytotoxicity medium. Then, in each tube, 0.1 mL of trypan blue solution (0.2% Trypan blue and sodium chloride NaCl solution (4:1)) was added and gently mixed. Lastly, 10 ul of tube 1 was used to load into a hemocytometer and determine cell counts. This was repeated for the other three sample tubes, and the resulting data was used to calculate the cytotoxicity of the experiment treatment in the data and results section. The entire procedure for cytotoxicity assay was condensed into a flowchart to allow for clearer perception (Figure 1).
Figure 1. Flow Chart Of Cytotoxicity Assay (6). The image shows a visual representation of the procedure used for the cytotoxicity assay.
Data And Results (1)
Table 1 shows the viable and non-viable cell counts in each 0.1 µl volume square in the hemacytometer and total call counts per thymus and spleen for the CD1 mouse. The cell viability for each treatment of cells in the tube was calculated in the last column of the table.
Table 1. Cell Counts In Hemacytometer For Thymus And Spleen(1)
Legend: Cyan highlights are calculated results; Tube 1, Thymus control; Tube 2, Thymus with anti-Thy AB; Tube 3, Spleen control; Tube 4, Spleen with anti-Thy AB; “E+” denotes exponential exponents of base 10;
The cyan-highlighted cell counts and percent fractions in Table 1 were calculated as follows.
Average per 0.1 µl viable count for tube 1 = ((881 / 0.1 µl) + (906 / 0.1 µl) + (897 / 0.1 µl) + (885 / 0.1 µl)) / 4 = 892 / 0.1 µl ,
average per 0.1 µl non-viable count for tube 1 = ((11 / 0.1 µl) + (16 / 0.1 µl) + (14 / 0.1 µl) + (13 / 0.1 µl)) / 4 = 14 / 0.1 µl ,
total cells per 0.1 µl count for tube 1 = 892 / 0.1 µl + 14 / 0.1 µl = 906 / 0.1 µl ,
total cells per thymus = 906 / 0.1 µl × 1000 µl/mL × 5 mL/thymus = 4.53 × 107 per thymus,
dead cells per thymus = 14 / 0.1 µl × 1000 µl/mL × 5 mL/thymus = 6.75 × 105 per thymus,
viable cells per thymus = 892 / 0.1 µl × 1000 µl/mL × 5 mL/thymus = 4.46 × 107 per thymus,
tube 1 cell viability = 4.46 × 107 / (4.53 × 107 ) × 100% = 98.5%.
Average per 0.1 µl viable count for tube 2 = ((203 / 0.1 µl) + (195 / 0.1 µl) + (168 / 0.1 µl) + (196 / 0.1 µl)) / 4 = 191 / 0.1 µl ,
average per 0.1 µl non-viable count for tube 2 = ((622 / 0.1 µl) + (558 / 0.1 µl) + (523 / 0.1 µl) + (561 / 0.1 µl)) / 4 = 566 / 0.1 µl ,
total cells per 0.1 µl count for tube 2 = 191 / 0.1 µl + 566 / 0.1 µl = 757 / 0.1 µl ,
total cells per thymus = 757 / 0.1 µl × 1000 µl/mL × 5 mL/thymus = 3.78 × 107 per thymus,
dead cells per thymus = 566 / 0.1 µl × 1000 µl/mL × 5 mL/thymus = 2.83 × 107 per thymus,
viable cells per thymus = 191 / 0.1 µl × 1000 µl/mL × 5 mL/thymus = 9.53 × 106 per thymus,
tube 2 cell viability = 9.53 × 106 / (3.78 × 107) × 100% = 25.2%.
Average per 0.1 µl viable count for tube 3 = ((1187 / 0.1 µl) + (1394 / 0.1 µl) + (1127 / 0.1 µl) + (1516 / 0.1 µl)) / 4 = 1306 / 0.1 µl ,
average per 0.1 µl non-viable count for tube 3 = ((132 / 0.1 µl) + (98 / 0.1 µl) + (177 / 0.1 µl) + (143 / 0.1 µl)) / 4 = 138 / 0.1 µl ,
total cells per 0.1 µl count for tube 3 = 1306 / 0.1 µl + 138 / 0.1 µl = 1444 / 0.1 µl ,
total cells per spleen = 1444 / 0.1 µl × 1000 µl/mL × 10 mL/spleen = 1.44 × 108 per spleen,
dead cells per spleen = 138 / 0.1 µl × 1000 µl/mL × 10 mL/spleen = 1.38 × 107 per spleen,
viable cells per spleen = 1306 / 0.1 µl × 1000 µl/mL × 10 mL/spleen = 1.31 × 108 per spleen,
tube 3 cell viability = 1.306 × 108 / (1.444 × 108) × 100% = 90.5%.
Average per 0.1 µl viable count for tube 4 = ((505 / 0.1 µl) + (486 / 0.1 µl) + (511 / 0.1 µl) + (515 / 0.1 µl)) / 4 = 504 / 0.1 µl ,
average per 0.1 µl non-viable count for tube 4 = ((144 / 0.1 µl) + (151 / 0.1 µl) + (142 / 0.1 µl) + (171 / 0.1 µl)) / 4 = 152 / 0.1 µl ,
total cells per 0.1 µl count for tube 4 = 504 / 0.1 µl + 152 / 0.1 µl = 656 / 0.1 µl ,
total cells per spleen = 656 / 0.1 µl × 1000 µl/mL × 10 mL/spleen = 6.56 × 107 per spleen,
dead cells per spleen = 152 / 0.1 µl × 1000 µl/mL × 10 mL/spleen = 1.52 × 107 per spleen,
viable cells per spleen = 504 / 0.1 µl × 1000 µl/mL × 10 mL/spleen = 5.04 × 107 per spleen,
tube 4 cell viability = 5.04 × 107 / (6.56 × 107) × 100% = 76.8%.
Table 2 shows the cytotoxicity of each treatment of cells for the CD1 mouse in the tube for both the thymus and spleen, the cytotoxicity index derived from cytotoxicity difference between treatment with Thy1.2 antibody and without, and the calculated T-cell counts. The percent T-cells for both the thymus and spleen were calculated in the last row of the table.
Table 2. Cytotoxicity Of Treatments Of Cells From Thymus And Spleen (1)
Legend: “E+” denotes exponential exponents of base 10; Ab, antibody
The equation used to calculate percent cytotoxicity was
(non-viable, dead cell count of thymus or spleen) / (total cell count) × 100%
, and the equation for the cytotoxicity index was
100(percent cytotoxcicity with antibody and complement) - (percent cytotoxicity with complement alone)100 - (percent cytotoxicity with complement alone)
For tube 1, the calculation of cytotoxicity was 6.75 × 105 / (4.53 × 107 ) × 100% = 1.5 % .
For tube 2, the calculation of cytotoxicity was 2.83 × 107 / (3.78 × 107 ) × 100% = 74.8 % .
For the thymus with antibody Thy1.2 and complement for the CD1 mouse, the cytotoxicity index was calculated as
100 × (74.8 - 1.5) / (100 - 1.5) = 73.3 .
For tube 3, the calculation of cytotoxicity was 1.38 × 107 / (1.44 × 108 ) × 100% = 9.5 % .
For tube 4, the calculation of cytotoxicity was 1.52 × 107 / (6.56 × 107 ) × 100% = 23.2 % .
For the spleen with antibody Thy1.2 and complement for the CD1 mouse, the cytotoxicity index was calculated as
100 × (23.2 - 9.5) / (100 - 9.5) = 15.1 .
The general equation used for the number of T cells was
number of T cells =
(number of dead cells with anti-Ty1.2 Ab) - (number of dead cells with complement alone) .
For the thymus, since the total number of cells in tube 1 and tube 2 were more than 10% different, the number of dead cells in tube 2, 566 / 0.1 µl was scaled by the total cell count ratio between tube 1 and 2 , 906/757, to obtain corresponding dead cells for tube 1 as 566 / 0.1 µl × 906/757 = 677 / 0.1 µl .
Number of T cells was calculated as
(677 / 0.1 µl - 14 / 0.1 µl) × 1000 µl/mL × 5 mL/thymus = 3.32 × 107 per thymus.
Percent of T cells was calculated as
3.32 × 107 / (4.53 × 107) × 100% = 73.3 % .
For the spleen, since the total number of cells in tube 3 and tube 4 were more than 10% different, the number of dead cells in tube 4, 152 / 0.1 µl was scaled by the total cell count ratio between tube 3 and 4 , 1444/656, to obtain corresponding dead cells for tube 1 as 152 / 0.1 µl * 1444/656 = 335 / 0.1 µl .
Number of T cells was calculated as
(335 / 0.1 µl - 138 / 0.1 µl) × 1000 µl/mL × 10 mL/spleen = 1.97 × 107 per spleen.
Percent of T cells was calculated as
1.97 × 107 / (1.444 × 108) × 100% = 13.6 % .
Discussion
Thy-1 (Thymocyte differentiation antigen 1), or CD90 is a thymocyte antigen and serves as a T-cell marker. In mice, there are two alleles for the Thy-1 gene, Thy1.1 (CD90.1) or Thy1.2 (CD90.2). The two alleles vary by one amino acid, with arginine in Thy-1.1 and glutamine in Thy-1.2. Thy1.2 (CD90.2), the antigen of interest in the experiment, is expressed on thymocytes, mature T-lymphocytes, epithelial cells, fibroblasts, brain cells, and hematopoietic cells. It is one of the most abundant glycoproteins on T-cells and is thought to play a role in processes such as lymphocyte function and apoptosis. The Thy1.2 version of the allele is most likely to be found in inbred mouse strains (7).
The complement system activation by immunoglobulin is vital in fighting pathogens in mammalian innate immune responses. The complement system generates the complement protein 3 (C3) convertase machinery to cleave C3 proteins on the surface of pathogen membranes and attach the cleaved C3b subunit onto the membrane, according to the textbook (3). As shown in Figure 2, the first step in activating the complement system in the classical pathway is for IgG or IgM to attach to the pathogen surface and complex with complement components 1q (C1q), 1r (C1r), and 1s (C1s) (3). Complement components 2 and 4 (C2 and C4, respectively) are cleaved by the IgG-C1q/C1r/C1s complex. As the reaction continues, once complement component 3b (C3b) is attached to the pathogen membrane, complement proteins 5, 6, 7, 8, and 9 (C5 through C9) are recruited and polymerized to form a membrane attack complex (MAC) in the pathogen membrane with a central pore. The cellular content of the pathogen subsequently leaks through the pore, killing the pathogen cell. There are 3 chemical pathways to generate the said C3 convertase: the classical, the alternative, and the lectin pathway. In the classical pathway, either the liver secretes C-reactive protein to bind to the pathogen membrane, or the antibodies immunoglobulin G or M (IgG or IgM, respectively) bind to the pathogen membrane as the first chemical step toward C3b attachment. In this experiment, the anti-Thy1.2 antibody was the IgG in the first chemical reaction step toward C3b attachment in the classical pathway.
Figure 2. Complement Activation Chemical Reactions (Gibson, J.)
Caption: C1 through C9, complement component 1 through 9; C3bBb, the alternative pathway’s C3 convertase; C4bC2b, the classical pathway’s C3 convertase that recruits a C3b protein to the pathogen membrane surface; MAC, membrane attack complex.
With complement cytotoxicity assay, this experiment aimed to verify the binding affinity of the anti-Thy1.2 antibodies to CD1 mouse T-cells and the fully functional pathway leading to MAC lysing of the cells. Thy1.2 is well known to be expressed by many mouse strains BALB/c, CBA/H-T6, CBA/J, CE, C3H/An, C3H/He, C3Hr/Bi, C57BL/6, C57BR, C57BR/cd, C57L, C58, DA, DBA/I, DBA/2, FL/2Re, GR/A, HRS, HSFS/N, but not all mouse strains, specifically, CD1 mouse strain was not verified in the literature (5). To prove the effectiveness of the anti-Thy1.2 antibodies against CD1 mice T-cells, the T-cell death counts of this experiment in the mouse organs should match the literature (4). The cell lysing in this experiment obtained the T-cell counts for the mouse thymus and spleen through the T-cell death counts.
This experiment successfully induced T-cell lyses with Thy1.2 antibodies and complement proteins C1 through C9, as shown in Table 1’s T-cell death counts. The percent of viable cells in the thymus decreased with the antibody and complement treatment. With only the complement treatment, 98.5% and 90.5% of cells from the thymus and the spleen were viable during cytometry counting, meaning that the complement-only solution did not induce significant cell lysis and that the cell extraction process did not damage most cells. In contrast, with the antibody and complement treatment combined, the viability of cells in both the thymus and the spleen dropped to 25.2% and 76.8%. This means that the treatment with anti-Thy1.2 antibody killed a large portion of cell populations.
Antibody Thy1.2 with complement activation had high cytotoxicity toward cells, as shown in Table 2’s calculation, 74.8% toward the thymus and 23.2% toward the spleen. The difference between the 74.8% and 23.2% cytotoxicities was expected as the thymus is the site of T-cell maturation, so a large population of immature T-cells and mature T-cells was released from the thymus tissue extract. When adjusted for preparation damages to cells by subtracting complement-only death counts of tube 1 and tube 3, the cytotoxicity index (C.I.) was obtained at 73.3% and 15.1% for the thymus and the spleen. Again, the large difference in the cytotoxicity between the organs was expected by the T-cell distribution difference, high concentration in the thymus, and lower concentration in the spleen.
With the expected, reasonable result of anti-Thy1.2 antibody toxicities with complement activation on 2 different organs, the T-cells killed by the experiment were calculated by subtracting the death count with complement-only reaction (tube 1 and tube 3 as the blank negative controls) from the death counts with antibody (tube 2 and tube 4). The number of T-cells in the thymus and the spleen were obtained as 3.32 × 107 per thymus and 1.97 × 107 per spleen. These numbers were close to literature descriptions, where T-cell populations in the mouse thymus were 9.7 × 107 double-positive and 8.8 × 107 pre-selection double-positive as the two largest T-cell categories with overlapping (4). The difference in absolute cell counts could be attributed to mouse specimen weights. The percentage of T-cells in the thymus cell populations was calculated as 73.3 % (Table 2), slightly lower than the literature descriptions of 97% and 100% (2, 7). The percentage of T-cells in the spleen cell populations was calculated as 13.6% (Table 2), slightly lower than the literature description of 35% and 23% (2, 7).
In conclusion, the experiment was a total success. The addition of Cedarlane’s anti-mouse CD-90.2 (Thy 1.2) monoclonal antibodies alongside complements successfully killed cells via complement activation, as attested by the increased cytotoxicity compared to the control. This was further attested by the cytotoxicity index of 73.3% for the thymus and 15.1% for the spleen. The thymus was found to have a larger percentage of T-cells than the spleen, which was expected due to the location of T-cell development. Overall, the experiment demonstrated the ability of anti-Thy1.2 antibodies to induce T-cell death via complement, and the results corresponded with the expected distribution of T-cells between the thymus and the spleen.
References
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Juris, S. J. (2022). Immunology. Oxford University Press.
Krueger, Andreas, Natalia Ziętara, and Marcin Łyszkiewicz. "T cell development by the numbers." Trends in immunology 38.2 (2017): 128-139.
Letarte, Michelle. "[57] Thy-1.1 and Thy-1.2 alloantigens: An overview." Methods in Enzymology 108 (1984): 642-653.
Royan, S.V. & A. Alton. (2024). Immunology Laboratory Manual Fall 2024. [Unpublished Immunology Laboratory Manual]. Department of Biological Sciences, University of Massachusetts, Lowell.
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