May 18, 2017
BiTE® T cell-dependent cellular cytotoxicity (TDCC) assay
- Sandra L. Ross1,
- Marika Sherman1,
- Patricia L. McElroy1,
- Julie A. Lofgren1,
- Gordon Moody1,
- Patrick A. Baeuerle1,
- Angela Coxon1,
- Tara Arvedson1
- 1Department of Oncology Research, Amgen Inc.
External link: https://doi.org/10.1371/journal.pone.0183390
Protocol Citation: Sandra L. Ross, Marika Sherman, Patricia L. McElroy, Julie A. Lofgren, Gordon Moody, Patrick A. Baeuerle, Angela Coxon, Tara Arvedson 2017. BiTE® T cell-dependent cellular cytotoxicity (TDCC) assay. protocols.io https://dx.doi.org/10.17504/protocols.io.hweb7be
Manuscript citation:
Ross SL, Sherman M, McElroy PL, Lofgren JA, Moody G, Baeuerle PA, Coxon A, Arvedson T, Bispecific T cell engager (BiTE) antibody constructs can mediate bystander tumor cell killing. PLoS ONE 12(8). doi: 10.1371/journal.pone.0183390
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: May 10, 2017
Last Modified: March 22, 2018
Protocol Integer ID: 5798
Abstract
T cell dependent cytotoxicity assay for measuring bispecific T cell engager (BiTE®) activity.
Overview
Overview
Two methods are described for measuring cytotoxicity – imaging (nuclear count) and CellTiter-Glo®. Set-up for the two assays are the same.
- Number of wells needed for each assay should be carefully calculated, depending on the number of cell lines, number of BiTE® antibody constructs, number of BiTE® dilutions, E:T ratios, controls and number of replicates.
- For 96-well plate assays, each well contains a final volume of 100 µl (10 µl 10X BiTE® dilutions), 40 µl T cells at desired E:T ratio and 50 µl target cells.
- In a typical 96-well assay, each well contains:
- 10,000 target cells
- 100,000 T cells/ well (10:1 E:T ratio)
- BiTE® dilutions starting at 100 pM final
- 2 columns of control wells (one with target cells, T cells and no BiTE® and the other with target cells only)
- Cytotoxicity is measured at 48h by imaging (nuclear count) and/or by luminescence (CellTiter-Glo)
- All steps are done in Biosafety laminar flow hood.
Assemble materials
Assemble materials
| Materials | Company | Cat.no. | |
| Sterile 96-well clear V-bottom polypropylene plates | Greiner | 651201 | |
| Flat-bottom white 96-well plates (CellTiter-Glo) | Corning | 3917 | |
| ViewPlate-96 black plate Packardview plate (imaging) | Perkin Elmer | 6005182 | |
| Effector cells – unstimulated pan T cells | AllCells | PB009-1F | |
| Target cells – SW620, NUGC4, MOLM13 | Amgen cell bank | - | |
| BiTE®s – EGFR, MEC14, CD33 | Amgen | - | |
| CellStripper™ Dissociation Reagent nonenzymatic 1X | Corning | 25056Cl | |
| 1x PBS | Gibco | 14190 | |
| 50 ml Falcon Tube | BD | 35 2070 | |
| 15 ml Falcon Tube | BD | 35 2096 | |
| Growth medium: RPMI 1640 medium Supplements: 100U/ml penicillin/streptomycin 10% heat-inactivated fetal bovine serum (FBS) | Gibco Gibco Gibco | 11875-093 15140-122 10082-147 | |
| Assay medium: RPMI 1640 medium Supplements: 1x nonessential amino acids (NEAA) 10mM HEPES 50µM 2-ß-mercaptoethanol 1mM sodium pyruvate 100U/ml penicillin/streptomycin 5% heat-inactivated fetal bovine serum (FBS) | Biochrom Gibco Gibco Sigma Gibco Gibco Gibco | FG1215 11140-050 15630-080 M6250 11360-070 15140-122 10082-147 | |
| CellTiter-Glo® | Promega | G7572-100ml G7571-10ml | |
| Hoechst 33342 nuclear dye | ThermoFisher | 62249 | |
| Formaldeyde 16% (w/v) methanol-free | Pierce/ThermoFisher | 28908 |
Thaw frozen T cells
Thaw frozen T cells
- Keep cells on dry ice till ready for use
- In a 37°C water bath, warm medium (Assay Media) which contains 5% FBS.
- Clean the frozen vial with 70% alcohol before thawing. In a biosafety hood, twist the cap a quarter-turn to relieve pressure, and then retighten the cap.
- Thaw cells in a 37°C water bath for approximately 2 mins. Do not remove the vial until cells are almost completely thawed with a small ice-crystal still visible.
- Clean the outside of the vial with EtOH. In a biosafety hood, measure the cell suspension volume.
- Transfer all the cells to 50ml Falcon tube.
- Slowly rinse the vial with 1 ml of pre-warmed media original cell vial to ensure complete retrieval.
- Slowly add, dropwise, the 1ml of media + cells into 50ml Falcon tube.
- Add 40 ml of media slowly and with swirling,
- Remove 0.5 ml cells for counting on ViCell (Beckman Coulter; default cell setting and 50 images).
- Centrifuge at 1,000 rpm for 10 mins at room temp.
- Remove supernatant.
- Gently resuspend cell pellet in Assay Medium. Add enough medium to bring up to approximate working dilution (generally best to add about 10-20% less medium than calculated so cells can be further diluted).
- Transfer to TC flask and put into 37 deg incubator until ready for use.
- Observe thawing procedure at: http://www.allcells.com/support/BioTube-How-to-thaw-cells.wmv
Prepare BiTE® dilutions
Prepare BiTE® dilutions
- Determine number of wells needed and make sure to have extra volume for dead volume on electronic pipets or liquid handling automation.
- Use deep well plate for BiTE® dilution (for larger volumes) or 96-well polypropylene plate (for smaller volumes.
- For 96-well plate: 10 BiTE® concentrations + two control wells (no BiTE®)
- Highest working dilution concentration (10X) = 1 nM, 3-fold dilutions X 9 dilutions
- Highest final concentration = 100 pM
- Dilute each BiTE® to 1 nM in Assay Media, then titrate 1:3 across plate in cols. 1-10; Assay Medium only in cols. 11-12.
- For example, add 300 µl 1nM BiTE® to col. 1 of plate and add 200 µl Assay Medium to cols. 2-12; remove 100 µl BiTE® from col. 1, add to col. 2 and mix; remove 100 µl BiTE® from col. 2, add to col. 3 and mix, and so on through col. 10.
- Volume each BiTE® concentration needed depends on number of cell lines and other parameters being tested and number of replicate wells or plates desired.
Prepare target cells
Prepare target cells
- Target cells are passaged 2-3X per week in Growth Medium and used for ≤15 passages before thawing a fresh vial.
- Determine number of wells needed and make sure to have extra volume for dead volume on electronic pipets or liquid handling automation.
- For suspension cells: Count cells and centrifuge needed number at 1,000 RPM or 300xg for 10 min; discard supernatant and wash once with Assay Media warmed to 37°C.
- For adherent cells: Use Cell Dissociation Solution (Sigma) to detach adherent cells, resuspend and count
- For 96-well plate assays, use 10,000 target cells/well
- Dilute cells to 200,000 cells/ml (10,000 cells/well, 50 µl/well)
Dilute T cells
Dilute T cells
- Determine number of wells needed and make sure to have extra volume for dead volume on electronic pipets or liquid handling automation
- Determine desired E:T ratio, e.g., 10:1 = 10 T cells for each target cell
- Dilute T cells prepared in step 2 above to 2.5X (see example below)
- For 10:1 E:T ratio with 10,000 target cells/well: 10X target cells = 100,000 T cells/well final; 2.5 X 100,000 = 2,500,000 cells/ml (2,500,000 cells/ml X 0.04 ml/well = 100,000 T cells/well)
Assay set-up
Assay set-up
- For imaging assay, use black, clear-bottom PackardView plates; for CellTiter-Glo assay, use white Corning plates (see Materials).
- Add 10 µl of (10X) BiTE® dilutions to wells in cols. 1-10; add 10 µl Assay Medium to wells in col. 11; add 50 µl Assay Medium to wells in col. 12
- Add 50 µl target cells to wells in all columns (e.g., 200,000cells/ml X 0.05ml = 10,000 cells/well)
- Add 40 µl T cells to wells in cols. 1-11 (e.g., 2,500,000 cells/ml X 0.04ml = 100,000 cells/well)
- Mixing occurs upon addition of cells to wells
- Leave assay plates at room temperature in hood for 30 min. before placing in tissue culture incubator
- Incubate at 37°C for 48 hours
Cytotoxicity Assay
Cytotoxicity Assay
- Imaging assay (this method is often combined with an immunofluorescence assay to measure e.g., target, ICAM-1, FAS such that both cytotoxicity and expression patterns can be measured in the same assays – see separate protocols)
- After incubation time, resuspend suspension cells in plate and remove with pipet (and save for subsequent assays) or vacuum manifold
- Wash plates 2 times with PBS: Add 100 µl/well PBS to remove any debris or T cells with quick rinse. Remove PBS with pipet or vacuum manifold. Repeat with second wash.
- In fume hood: Remove PBS and fix with 4% methanol-free formaldehyde in PBS, 15 min at room temperature (100 µl/well); remove formaldehyde and dispose in appropriate chemical waste; add back 100 µl/well PBS
- Remove PBS and add 100 µl/well Hoechst nuclear dye (5 µg/ml in PBS); incubate for 30 min. at room temperature
- Remove Hoechst dye and dispose in appropriate chemical wasted; add back add back 100 µl/well PBS
- Read on ArrayScan™ using Target Activation bioapplication optimized for cell line in use. Count cells in 16 10X fields. Note: Any remaining T cells left in wells after washes are excluded from cell counts by the lower nuclear size threshold.
- CellTiter-Glo® assay
- After incubation time, resuspend suspension cells in plate and remove with pipet (and save for subsequent assays) or vacuum manifold
- Wash plates 2 times: Add 100µl/well Assay Medium to remove any debris or T cells with quick rinse. Remove media with pipet or vacuum manifold. Repeat with second wash.
- Add 100µl/well of room temperature Assay Medium and add 100uL of room temperature Cell CellTiter-Glo® reagent
- Incubate 10 minutes.
- Read on luminometer.
Analysis of supernatants and T cells removed from cytotoxicity assay plates
Analysis of supernatants and T cells removed from cytotoxicity assay plates
- Crude supernatants (containing soluble factors and T cells) can be used directly in additional cytotoxicity assays, or used in Transwell® assays (see Transwell® assay protocol); alternatively soluble factors and T cells can be separated and analysed independently.
- Carefully remove supernatants (containing soluble factors and T cells) from wells into 96-well V-bottom polypropylene plates and centrifuge at 300 x g for 5 min
- Test for soluble factors: Collect cell-free medium and test for cytokines by MSD or EIA according to Manufactures’ instructions (medium can be stored at -80°C prior to testing). Please see additional protocols for cytokine detection.
- Test for T cell activation: After removing cell-free medium (above), T cells are tested by flow cytometry for T cell activation markers (see T cell activation protocol)