compensation
DESCRIPTION
COMPENSATION. FL1. FITC. +. detector. FITC -. PE. gain=1. -. FL2-%FL1. FITC. FL2. PE. +. PE -. FITC. gain=1. detector. -. COMPENSATION. FL1. FITC. +. detector. FITC -. PE. gain=1. -. PE. FL2. PE. +. PE -. FITC. gain=1. detector. FL1-%FL2. -. - PowerPoint PPT PresentationTRANSCRIPT
COMPENSATION
FITC
FITC - PE
PE - FITC
-
-
+
+gain=1
gain=1detector
detectorPE
FITC
FL1
FL2
FL2-%FL1
COMPENSATION
FITC
FITC - PE
PE - FITC
-
-
+
+gain=1
gain=1detector
detectorPE
PE
FL1
FL2
FL1-%FL2
Uncompensated vs Compensated
FL1
FL2
FL1
FL2
COMPENSATION IS INTENSITY DEPENDENT
uncompensated partially compensated
fully compensated
COMPENSATE INSTRUMENT USING STAINED CELLS
1. Adjust PMT voltages using unstained cells
2. Adjust compensation for each fluorochrome
0 256 512 768 1024
030695001FSC ->
0 256 512 768 1024
030695001FSC ->
Lymphogated Cells for Four Color Single Laser Compensation
A B
FSC FSC
SS
C
SS
C
10 10 10 10 100 1 2 3 4
042095001FL1 ->
10 10 10 10 100 1 2 3 4
042095001FL4 ->
10 10 10 10 100 1 2 3 4
042095001FL3 ->
10 10 10 10 100 1 2 3 4
042095001FL1 ->
10 10 10 10 100 1 2 3 4
042095001FL3 ->
10 10 10 10 100 1 2 3 4
042095001FL3 ->
FITC
FITC
PE-TxRd PE-Cy5 or PerCP
PE-Cy5 or PerCP PE-Cy5 or PerCP
PE
PE
PE
PE-TxRd
PE-TxRd
FITC
Setting the High Voltage
10 10 10 10 100 1 2 3 4
042095002FL1 ->
10 10 10 10 100 1 2 3 4
042095002FL4 ->
10 10 10 10 100 1 2 3 4
042095002FL3 ->
10 10 10 10 100 1 2 3 4
042095002FL1 ->
10 10 10 10 100 1 2 3 4
042095002FL3 ->
10 10 10 10 100 1 2 3 4
042095002FL3 ->
Compensation adjustment using FITC stained lymphocytes
PE
PE
PE
PE-TxRd
PE-TxRd
FITC-CD45
FITC-CD45
PE-TxRd PE-Cy5 or PerCP
PE-Cy5 or PerCP PE-Cy5 or PerCP
FITC-CD45
10 10 10 10 100 1 2 3 4
042095003FL1 ->
10 10 10 10 100 1 2 3 4
042095003FL4 ->
10 10 10 10 100 1 2 3 4
042095003FL3 ->
10 10 10 10 100 1 2 3 4
042095003FL1 ->
10 10 10 10 100 1 2 3 4
042095003FL3 ->
10 10 10 10 100 1 2 3 4
042095003FL3 ->
Compensation adjustment using PE stained lymphocytes
FITC PE-TxRd PE-Cy5 or PerCP
FITC PE-Cy5 or PerCP PE-Cy5 or PerCP
PE-CD4
PE-CD4
PE-CD4
PE-TxRd
PE-TxRd
FITC
10 10 10 10 100 1 2 3 4
042095004FL1 ->
10 10 10 10 100 1 2 3 4
042095004FL4 ->
10 10 10 10 100 1 2 3 4
042095004FL3 ->
10 10 10 10 100 1 2 3 4
042095004FL1 ->
10 10 10 10 100 1 2 3 4
042095004FL3 ->
10 10 10 10 100 1 2 3 4
042095004FL3 ->
Compensation adjustment using PE-TxRd stained lymphocytes
PE
PE
PE
ECD-CD4
FITC
FITC
ECD-CD4 PE-Cy5 or PerCP
PE-Cy5 or PerCP PE-Cy5 or PerCP
FITC
ECD-CD4
10 10 10 10 100 1 2 3 4
042095005FL1 ->
10 10 10 10 100 1 2 3 4
042095005FL4 ->
10 10 10 10 100 1 2 3 4
042095005FL3 ->
10 10 10 10 100 1 2 3 4
042095005FL1 ->
10 10 10 10 100 1 2 3 4
042095005FL3 ->
10 10 10 10 100 1 2 3 4
042095005FL3 ->
Compensation adjustment using PE-CY5 stained lymphocytes
PE
PE
PE
FITC
FITC TC-CD45TC-CD45
TC-CD45
FITC
PE-TxRd
PE-TxRd
PE-TxRd
10 20 30 40 50 60 70 80 90 100 120
FSC -->
1020
3040
5060
7080
90100
120
SS
C -->
10 1 10 2 10 3 10 4
FL1 -->
101
102
103
104
FL
2 --> R1
1 2
3 4
FSC
SS
C
R1
R2 R3
R4
FITC-CD45
PE-C
D4
10 1 10 2 10 3 10 4
FL4 -->
10
110
210
310
4
FL
2 --> R9
9 10
11 12
PE-CY5-CD8
PE-C
D4
R4 R5
R6
10 1 10 2 10 3 10 4
FL1 -->
101
102
103
104
FL
3 --> R13
13 14
15 16
PE-CY5-CD8
AP
C-C
D8
R8
R9
R10
COMPENSATION UP TO FOUR COLORS WITH TWO LASERS
10 10 10 10
FITC-CD3
PE
-CD
8
10
TC
-CD
2
FITC-CD3
PE
-CD
8
TC-CD22 10 10
10 2 10 4
EC
D-C
D4
FITC-CD310 10 10 10
EC
D-C
D4
TC-CD2
10 2 10 10
PE
-CD
8
ECD-CD4
UNCOMPENSATED
SOFTWARE COMPENSATION
101 102 103 104
10
1
10
2
10
3
10
4
PE
CD
8 -
->
PE
-CD
8
TC-CD2101 102 103 104
10
1
10
2
10
3
10
4
PE
-CD
8
ECD-CD4
101 102 103 104
10
1
10
2
10
3
10
4
TC
-CD
2
FITC-CD3101 102 103 104
10
1
10
2
10
3
10
4
EC
D C
D4 -
->
EC
D-C
D4
FITC-CD3101 102 103 104
10
1
10
2
10
3
10
4
EC
D-C
D4
TC-CD2
101 102 103 104
10
1
10
2
10
3
10
4
PE
-CD
8
FITC-CD3
R1
R2
R3
R4
R5
R6
101 102 103 104
FITC CD3 -->
10
1
10
2
10
3
10
4
PE
CD
8 -
->
PE
-CD
8
FITC-CD3101 102 103 104
10
1
10
2
10
3
10
4
PE
CD
8 -
->
PE
-CD
8
TC-CD2
INSTRUMENT COMPENSATION
101 102 103 104
ECD CD4 -->
10
1
10
2
10
3
10
4
PE
CD
8 -
->
PE
-CD
8
ECD-CD4
101 102 103 104
10
1
10
2
10
3
10
4
TC C
D2
-->
TC
-CD
2
FITC-CD3101 102 103 104
10
1
10
2
10
3
10
4
EC
D C
D4
-->
EC
D-C
D4
FITC-CD3101 102 103 104
10
1
10
2
10
3
10
4
EC
D-C
D4
TC-CD2
PE, TX-RED & PE-CY5COMPENSATION
TC CD2 -->101 102 103 10
10
1
10
2
10
3
10
PE
-CD
8
TC-CD2101 102 103 104
ECD CD4 -->1
0
1
10
2
10
3
10
4
PE
-CD
8
ECD-CD4
POOR
GOOD
101 102 103 104
10
1
10
2
10
3
10
4
PE
-CD
8
TC-CD2101 102 103 104
ECD CD4 -->
10
1
10
2
10
3
10
4
PE
-CD
8
ECD-CD4
R3
R3
A C
B D