Cytotoxicity LDH Assay Kit-WST

Transcription

1 ytotoxicity L ssay Kit-WST Notice to Users This is a supporting manual for antibody-dependent cell-mediated cytotoxicity () assay. or the contents of this kit and the preparation procedure of Working Solution, please see the technical manual originally attached to the ytotoxicity L ssay Kit-WST. or ccurate Measurement L activity can be different depending on the cell type. Therefore, when cell type is changed, please confirm optimum cell number of target cells before assay. Optimization of cell number assay Selection of the ssay There are two methods: homogeneous assay (Page 1) and non-homogeneous assay (Page 4), please select the assay method according to your experiments. omogeneous ssay Optimization of ell Number 1. fter washing target cells with the medium, prepare cell suspension to 5 x 10 5 cells/ml in the medium. 2. dd 100 μl of the medium to each well of 96-well plates. 3. In the Row of 96-well plate (triplicates of TMR(igh ontrol) and TSR (Low ontrol)) add 100 μl of target cells prepared in Step 1. and mix by pipetting (2.5 x 10 4 cells/well). Next, take 100 μl from the Row and add it to the Row. Repeat the serial dilution process as indicated in igure 1. TMR (Target Maximum Release, igh ontrol) Lysis uffer is added to the target cells L released from all of the cells. 1 2 TMR TSR ,000 cells/well 25,000 cells/well TSR (Target Spontaneous Release, Low ontrol) Only medium is added to the target cells Spontaneous L released from the cells. M (ulture Medium ackground, ackground ontrol) L activity contained in the medium 12,500 cells/well 6,250 cells/well 3,125 cells/well 1,563 cells/well 12,500 cells/well 6,250 cells/well 3,125 cells/well 1,563 cells/well M 0 cells/well igure 1 Plate arrangement 4. Incubate the plate at 37 o in O 2 incubator. *Use the same incubation time as the assay. 5. dd 10 μl of Lysis uffer into igh ontrol (TMR) wells. 6. Incubate the plate at 37 o for 30 minutes in O 2 incubator. 7. dd 100 μl of Working Solution to all of the wells. Protect the plate from light and incubate it at the room temperature for 30 minutes. *uration of the color reaction should be optimized based on the target cell types. 8. dd 50 μl of Stop Solution to all of the wells. 9. Measure the absorbance at 490 nm by a microplate reader. 10. Set target cell number per well which satisfies either of below conditions ) The difference of TMR and TSR absorbance (O TMR - O TSR ) 2 O M ) bsorbance difference between TMR and TSR at the maximum ) The absorbance of TMR (O TMR ) at above 1.0 below Issued July 31, 2017

2 (omogeneous assay protocol) Preparation of ntibody Solution Prepare 10 different concentration of antibody solution including zero concentration of antibody solution. ntibody Solution should be diluted using the medium. ssay Please refer to the plate arrangement (igure 2) and the amount of solution in each well (Table 1) efinition of the Wells R (xperimental Release): L released when all antibody solution, effector cells and target cells are mixed SR (ffector Spontaneous Release): spontaneous release of L by effector cells TSR (Target Spontaneous Release): spontaneous release of L by target cells TMR (Target Maximum Release): L released from the cells after the addition of Lysis buffer to the target cells M (ulture Medium ackground): L contained in the medium V (Volume orrection ontrol): L from addition of Lysis uffer in the medium, used for volume correction M R1 R2 R3 R4 R5 R6 R7 R8 R9 R0 TSR TMR R1' R2' R3' R4' R5' R6' R7' R8' R9' R0' SR V R wells can be used for different type of test substance. It is recommended to add medium in the Row and including M wells. igure 2 Plate arrangement Table 1 mount of solution in each well (omogeneous ssay) 2

3 (omogeneous assay) eneral Protocol 1. dd each different concentration antibody solution into R wells. 2. dd Target ell solution into R, TMR, and TSR wells. 3. dd ffector ell solution into R and SR wells. 4. dd Medium into SR, TSR, TMR, M, and V wells. 5. entrifuge at 250 x g for 4 minutes. 6. Incubate 37 o in O 2 incubator for an appropriate time. 7. dd Lysis buffer to TMR and V wells. 8. Incubate at 37 o in O 2 incubator for 30 minutes. 9. dd 100 μl of working solution to all of the well. 10. Protect the plate from light, and incubate it at room temperature for 30 minutes. 11. dd 50 μl of stop solution to all of the wells. alculation of ytotxicity 12. Measure the absorbance at 490 nm. 1. Subtract absorbance of M from absorbance of R, SR, TSR wells. 2. Subtract absorbance of V from the absorbance of TMR wells. 3. Plot the data into the following equation to obtain the cytotoxicity (%). ytotoxicity(%) = R - SR - TSR TMR - TSR 100 R: SR: TSR: TMR: O R O SR O TSR O TMR - O V xperimental xample 50 ssay using monolonal antibody erceptin omogenous ssay 40 ytotoxicity 細胞傷害率 (%) (%) Test Substance: Target ell (T ell): ffector ell ( ell): Medium: Ratio of ell and T ell: erceptin 濃度 (μg/ml) erceptin onc. (ug/ml) Measured two times with the same condition(red, lue) erceptin SK-R-3 (1x10 4 cells/well) PM (1x10 5 cells/well) RPMI1640 (2% S, 1% ntibiotic-antimicotic) 10:1 3

4 Non-omogeneous ssay Optimization of ell Number 1. fter washing target cells with the medium, prepare cell suspension to 5 x 10 5 cells/ml in the medium. 2. dd 100 μl of the medium to each well of 96-well plates. 3. In the Row of 96-well plate (triplicates of TMR(igh ontrol) and TSR (Low ontrol)) add 100 μl of target cells medium prepared in Step 1. and mix by pipetting (2.5 x 10 4 cells/well). Next, take 100 μl from the Row and add it to the Row. Repeat the serial dilution process as indicated in igure 3. TMR (Target Maximum Release, igh ontrol) Lysis uffer is added to the target cells L released from all of the cells. TSR (Target Spontaneous Release, Low ontrol) Only medium is added to the target cells Spontaneous L released from the cells. M (ulture Medium ackground, ackground ontrol) L activity contained in the medium 1 2 TMR TSR ,000 cells/well 25,000 cells/well 12,500 cells/well 12,500 cells/well 6,250 cells/well 6,250 cells/well 3,125 cells/well 3,125 cells/well 1,563 cells/well 1,563 cells/well M 0 cells/well igure 3 Plate arrangement 4. dd 100 μl medium to all of the wells. 5. Incubate the plate at 37 o in O 2 incubator *Use the same incubation time as the assay. 6. dd 20 μl of Lysis uffer into igh ontrol (TMR) wells. 7. Incubate the plate at 37 o for 30 minutes in O 2 incubator 8. entrifuge at 250 x g for 4 minutes. 9. Remove 100 μl of supernatant from all the wells, and add it to a new 96 well plates for measurement 10. dd 100 μl of Working Solution to all of the wells. Protect the plate from light and incubate it at the room temperature for 30 minutes. *uration of the color reaction should be optimized based on the target cell types. 11. dd 50 μl of Stop Solution to all of the wells. 12. Measure the absorbance at 490 nm by a microplate reader. 13. Set target cell number per well which satisfies either of below conditions ) The difference of TMR and TSR absorbance (O TMR - O TSR ) 2 O M ) bsorbance difference between TMR and TSR at the maximum ) The absorbance of TMR (O TMR ) at above 1.0 below 3.0 Preparation of ntibody Solution Prepare 10 different concentration of antibody solution including zero concentration of antibody solution. ntibody Solution should be diluted using the medium. ssay Please refer to the plate arrangement (igure 3) and the amount of solution in each well (Table 2) efinition of the Wells R (xperimental Release): L released when all antibody solution, effector cells and target cells are mixed SR (ffector Spontaneous Release): spontaneous release of L by effector cells TSR (Target Spontaneous Release): spontaneous release of L by target cells TMR (Target Maximum Release): L released from the cells after the addition of Lysis buffer to the target cells M (ulture Medium ackground): L contained in the medium V (Volume orrection ontrol): L from addition of Lysis uffer in the medium, used for volume correction 4

5 (Non-omogeneous ssay) M R1 R2 R3 R4 R5 R6 R7 R8 R9 R0 TSR TMR R1' R2' R3' R4' R5' R6' R7' R8' R9' R0' SR V R wells can be used for different type of test substance. It is recommended to add medium in the Row and including M wells. igure 4 Plate arrangement Table 2 mount of solution in each well (Non-homogeneous ssay) eneral Protocol 1. dd each different concentration antibody solution into R wells. 2. dd Target ell solution into R, TMR, and TSR wells. 3. dd ffector ell solution into R and SR wells. 4. dd Medium into SR, TSR, TMR, M, and V wells. 5. entrifuge at 250 x g for 4 minutes. 6. Incubate at 37 o in O 2 incubator for an appropriate time. 7. dd Lysis buffer to TMR and V wells. 8. Incubate at 37 o in O 2 incubator for 30 minutes. 9. entrifuge at 250 x g for 4 minutes. 10. Remove 100 μl from each well and add it to a new 96-well plate. 11. dd 100 μl of working solution to all of the well. 12. Protect the plate from light, and incubate it at room temperature for 30 minutes. 13. dd 50 μl of stop solution to all of the wells. 14. Measure the absorbance at 490 nm. 5

6 (Non-omogeneous ssay) alculation of ytotoxicity 1. Subtract absorbance of M from absorbance of R, SR, TSR wells. 2. Subtract absorbance of V from the absorbance of TMR wells. 3. Plot the data into the following equation to obtain the cytotoxicity (%). ytotoxicity (%) = R - SR - TSR TMR - TSR 100 R: SR: TSR: TMR: O R O SR O TSR O TMR - O V xperimental xample 50 ssay using monolonal antibody erceptin Non-omogeneous ssay 40 ytotoxicity 細胞傷害率 (%) (%) erceptin onc. 濃度 (μg/ml) (ug/ml) * Measured two times with the same condition(red, lue) Test Substance: Target ell (T ell): ffector ell ( ell): Medium: Ratio of ell and T ell: erceptin SK-R-3 (1x10 4 cells/well) PM (1x10 5 cells/well) RPMI1640 (2% S, 1% ntibiotic-antimicotic) 10:1 If you need more information, please contact ojindo technical service. ojindo Molecular Technologies, Inc. 30 West ude r., Suite 260, Rockville, M 20850, US Toll free: Phone: ax: mail: info@dojindo.com Web: 6