Immunological techniques

3.3.1 Isolation of mouse splenocytes

Mice (BL6-J and DBA2J) were sacrificed by cervical dislocation and then dissected under sterile conditions. Spleens were collected in a dish containing 1X PBS, a 70 µm filter was placed into 50 ml polypropylene tube and rinsed with RPMI medium. The spleen was then transferred on the filter. With the help of piston of a 5 ml syringe the spleen was pressed against the filter to dissociate the cells and continuously rinsed with medium.

RBC lysis was done by incubating the splenocytes with 1X RBC lysis buffer (Biolegend) for 5 min at room temperature. The remaining splenocytes were then washed and resuspended in 10% RPMI and counted in a Neubauer chamber. These splenocytes were used for the co- culture with PSC-derived cells.

3.3.2 Immune cell proliferation assay

The influence of PSC-derived cells on the activation or proliferation of splenocytes and T-lymphocytes was analyzed using a co-culture approach. 1 x 10⁵ PSC-derived cells, in the form of EBs were transferred into suspension culture 48-well cell culture plates.

Splenocytes were washed with PBS twice to remove serum. The cells were than resuspended at double the desired final concentration in PBS (pre-warmed to room temperature).10 µmol/L solution of Cell Proliferation Dye eFluor® 670 (ebioscience) in PBS (pre-warmed to room temperature) was prepared. This was mixed with the 2X cell suspension at 1:1 ratio and incubated for 10 minutes at 37°C in the dark. The eFluor®

670 dye (Appendix 2) labeling was stopped by adding 4-5 volumes of cold 10% RPMI

42 and incubated on ice for 5 minutes. The cells were then washed 3 times with 10%

RPMI.

1 x 10⁶ eFluor® 670 labeled splenocytes were then added to 1 x 10⁵ PSC-derived cells (non-myocytes/cardiomyocytes; NM/CM) and cultured for 4 days with 10% RPMI at 37

°C and 5% CO2. We used two different conditions: PSC-derivatives co cultured with DBA splenocytes (MHC-match for H-2^d/d locus) and PSC-derivatives co cultured with BL6 splenocytes (H-2^b/b, MHC-mismatch). Labeled splenocytes treated with 5 µg/mL concanavalin A (Con.A) served as positive control. Unlabelled splenocytes treated with Con.A served as negative control. Similarly EHMs made with PSC-CM (H-2^d/d) and DBA MEF (H-2^d/d) were incubated with DBA (H-2^d/d) and BL6 (H-2^b/b) labeled splenocytes for 4 days on a shaker with RPMI medium (Appendix 2) at 37°C and 5% CO2. Also EHMs made with PSC-CM (H-2^d/d) and BL6-MEF (H-2^b/b) were treated with DBA (H-2^d/d) and BL6 (H-2^b/b) splenocytes in the same way. A typical experimental set up is shown in Figure 9.

Figure 9: Experimental setup for in vitro proliferation assay. Different conditions were tested in a 48 well plate. Con.A: concanavalin A, NM: non-myocytes, CM: cardiommyocytes, Spl: splenocytes.

After 4 days of co-culture splenocytes were collected and filtered through 70 µm cell strainer to remove the EBs. The strained splenocytes were than stained with Alexa 488

Materials and Methods

43 labelled CD3 (Biolegend) and the proliferation of CD3 positive splenocytes was analyzed using flow cytometry.

3.3.3 Isolation of T-lymphocytes from spleen

Splenocytes were isolated from spleen as mentioned above. T-lymphocytes were isolated from splenocytes by using the mouse Pan T cell isolation kit II (Miltenyi biotec 130-095-130). 10⁷ splenocytes were resuspended in 40 µl of buffer(containing PBS, pH 7.2, 0.5% BSA, and 2 mmol/L EDTA).10 µl of Biotin-Antibody Cocktail (Cocktail of biotin-conjugated monoclonal antibodies against CD11b, CD11c, CD19, CD45R [B220], CD49b [DX5], CD105, Anti-MHC class II, and Ter-119) was added to 10⁷ total cells. The cell suspension was mixed well and incubated for 5 minutes in the refrigerator (4°C) followed by the addition of 30 µl of buffer per 10⁷ total cells. Finally 20 µl of anti-biotin microbeads per 10⁷ total cells was added and mixed well. This cell suspension was incubated for 10 minutes at 4°C followed by magnetic separation on LS columns mounted on a quadro MACS separator (Miltenyi Biotec). A minimum of 500 µl cell suspension was added to LS columns, the flow-through containing unlabeled cells was collected, representing the enriched T-cells. The column was washed with 3 ml of buffer and the pass through was collected representing the enriched T-cells. The cells were counted using a Neubauer chamber. T-cell purity was measured by flow cytometry.

3.3.4 In vitro cytotoxicity assay

To assess cytotoxic effects of T-cells cardiomyocytes were obtained from the spinner flask cultures dissociated as mentioned above, and seeded on gelatin coated 96 well plates at a concentration of 10⁵ cells per well. T-lymphocytes were isolated from the spleens of BL6-J and DBA2J mice as mentioned above and added to the cardiomyocytes at a concentration of 10⁶ cells per well (CM: T-cells ratio, 1:10).The co-culture was incubated at 37°C and 5% CO2 for 4 hrs and proceeded with the measurement of cell lysis by using the cytotox 96^® non-radioactive cytotoxixity assay kit (promega).

44 Briefly 100 µl of 10% RPMI medium was added to all the wells of a 96 well plate. For maximum lysis, 10 µl of lysis solution per 100 µl of medium was added and incubated at 37°C for 45 minutes. The plate was than centrifuged at 250 x g for 4 minutes. Then 50 µl of supernatant from all wells was transferred into a fresh 96-well flat-bottom (enzymatic assay) plate followed by the addition of 50 µl of substrate mix to each well of the enzymatic assay plate. The plate was then covered with foil or an opaque box to protect it from light and incubated for 30 minutes at room temperature. Finally 50 µl of stop solution was added to each well and the absorbance was recorded at 490 nm or 492 nm with the help of 96 well plate reader (Flexstation 3, molecular devices).

Percent cytotoxicity was measured by using the following formula:

% Cytotoxicity = Experimental – Effector Spontaneous – Target Spontaneous x 100 Target Maximum – Target Spontaneous

The average absorbance values of the culture medium background were subtracted from the average absorbance values of experimental, target spontaneous and effector spontaneous. Similarly the average absorbance values of volume correction control were subtracted from average absorbance values of target maximum.