1ml per sample. The samples were incubated at 4⁰C overnight. The following day, the samples were washed with DPBS at 4⁰C and then washed with 2ml 1 perm buffer twice. Anti-mouse Foxp3- PE Ab was added in a 1:100 dilution for 30 mins at 4⁰C. The samples were washed with perm buffer and resuspend in DPBS for flow cytometry analysis.
Table 2.4 Antibody panel for SHPS-1 Phenotyping
Panel PerCP FITC APC PE PB
DC 1 CD45 CD11b CD11c CD103 B220
DC 2 CD4 CD11b CD11c CD8 B220
Treg CD3 CD25 FoxP3 CD4 2.2.1.2.5 Analysis of Cell Populations in Allo- and Xeno-transplantation Recipients
The spleen, ALN and DLN were harvested from mice at certain time points post allo or xeno-transplantations. SHPS-1 and WT mice who were the recipients of skin allo-transplants, DEREG, WT and Rag-/- mice who were the recipients of NICC xeno-transplants were harvested for flow cytometry analysis. The procedure for isolation of splenocytes is described in 2.2.1.1.2, then staining protocols were followed as per the same steps as 2.2.1.2.4. Splenocytes and lymphocytes were stained with anti-mouse-CD4-PerCP, …show more content…
To normalise islet volume, the islet equivalent (IEQ) was calculated from the diameters, assuming that 1 IEQ equals to a spherical islet with a diameter of 150 μm. The islets under each diameter category was then converted to IEQ using a set of islet factors, and the sum was calculated to obtain the total IEQ number for each islet preparation (Table 2.5).
Table 2.5 Islet factors for IEQ calculation.
ISLET DIAMETER (µm) MEAN VOL (µm3) IEQ: Conversion into islets of 150um diameter
Islet number (n) x islet factor
50-100 294,525 n / 6.00
100-150 1,145,373 n / 1.50
150-200 2,977,968 n x 1.7
200-250 6,185,010 n x 3.5
250-300 11,159,198 n x 6.3
300-350 18,293,231 n x 10.4
350-400 27,979,808 n x
Table 2.4 Antibody panel for SHPS-1 Phenotyping
Panel PerCP FITC APC PE PB
DC 1 CD45 CD11b CD11c CD103 B220
DC 2 CD4 CD11b CD11c CD8 B220
Treg CD3 CD25 FoxP3 CD4 2.2.1.2.5 Analysis of Cell Populations in Allo- and Xeno-transplantation Recipients
The spleen, ALN and DLN were harvested from mice at certain time points post allo or xeno-transplantations. SHPS-1 and WT mice who were the recipients of skin allo-transplants, DEREG, WT and Rag-/- mice who were the recipients of NICC xeno-transplants were harvested for flow cytometry analysis. The procedure for isolation of splenocytes is described in 2.2.1.1.2, then staining protocols were followed as per the same steps as 2.2.1.2.4. Splenocytes and lymphocytes were stained with anti-mouse-CD4-PerCP, …show more content…
To normalise islet volume, the islet equivalent (IEQ) was calculated from the diameters, assuming that 1 IEQ equals to a spherical islet with a diameter of 150 μm. The islets under each diameter category was then converted to IEQ using a set of islet factors, and the sum was calculated to obtain the total IEQ number for each islet preparation (Table 2.5).
Table 2.5 Islet factors for IEQ calculation.
ISLET DIAMETER (µm) MEAN VOL (µm3) IEQ: Conversion into islets of 150um diameter
Islet number (n) x islet factor
50-100 294,525 n / 6.00
100-150 1,145,373 n / 1.50
150-200 2,977,968 n x 1.7
200-250 6,185,010 n x 3.5
250-300 11,159,198 n x 6.3
300-350 18,293,231 n x 10.4
350-400 27,979,808 n x