Purpose | Measure antibody-dependent NK92 cell-mediated cytotoxicity using calcein AM-stained MDA-MB-231 |
Existing Method(s) | LDH Release Assay |
Target Cell Type | Target: MDA-MB-231; Effector: NK92 cells |
Experiment Plan | Scan plate using Bright field and Green Fluorescent channel |
Hypothesis | By measuring the changes in the number of calcein AM-positive cells over time, the % cytotoxicity can be calculated using time 0 and control for normalization |
Celigo Setup
Plate Type | Greiner 655090 96-well black wall clear bottom |
Scan Channels | Bright field and Green |
Resolution | 1 µm/pixel |
Scan Area | Whole well |
Analysis Method | Target 1 + 2 |
Scan Frequency | Every 2 hours, up to 6 hours |
Scan Time | ~4 min |
Assay Protocol and Plate Setup
Goal:
Measure ADCC of NK92 cell-mediated cytotoxicity using calcein AM-stained MDA-MB-231 for 6 hours
Protocol:
Cell preparation
- MDA-MB-231 were obtained from ATCC and cultured in RPMI 1640 media
- NK92-CD16 cells were purchased and expanded in RPMI 1640 media
- MDA-MB-231 cells were stained with 10 µM of calcein AM (Nexcelom, Cat# CS1-0119) for 30 min and then washed 3 times with RPMI media
- The cells were then seeded into the 96-well plate at 10,000 cells/well
- Next, NK92 cells were added at 5:1 E:T ratio 6. Finally, antibodies were added at 0, 1, 10, 100 pg/ml; 1, 10, 100 ng/ml; 1 µg/ml; and control antibody
- The spontaneous release samples were stained Target cells without Effector cells
Data Collection
- After adding the cells and antibodies, the plate was centrifuged to settle the cells to the bottom
- Immediately after, the plate was scanned in Celigo using Target 1 (Green) + 2 (BF) for t = 0 h
- Repeated the scanning for t = 2, 4, and 6 h
Data Analysis
- The images for each time point were analyzed to count total number of calcein AM-positive cells in each well
- Used the last time point as a baseline for setting up the gating parameters
- Made sure only the bright calcein AM cells were counted o The pieces of bright debris were gated out o The dim cells were gated out
Data Calculation
- The calcein AM-positive Target cells co-cultured with and without Effector cells were counted and recorded
- The calcein AM-positive Target cells with and without antibodies were counted and recorded
- The calcein AM-positive Target cells only were counted and recorded
- Normalized to t = 0
- Normalized to spontaneous release
% 𝐶𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦 = % 𝑐𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦 (𝑠𝑎𝑚𝑝𝑙𝑒) − % 𝑐𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦 (𝑠𝑝𝑜𝑛𝑡) - The % cytotoxicity was calculated for every well and averaged to generate dose-response and time course monitoring
Results
1. Celigo-captured calcein AM fluorescent images at different time points
- Below is an example of plate view in the results section showing cells counted on the plate
- Below are example calcein AM images for MDA-MB-231 at different time points
- The number of calcein AM-positive cells decreased as time increased
- The purple outlines indicate the counted calcein AM-positive cells
- Dose-dependent cytotoxicity results at different time points
- The results showed increasing % cytotoxicity as the antibody concentration increased for each time point
2. Immune complex formation
- Below are example calcein AM and bright field overlay images, which showed the formation of immune complex clusters at different times
- Below are example calcein AM and bright field overlay images at 6 hours, which showed the formation of immune complex clusters at different antibody dosages
Conclusion
- The Celigo was able to measure ADCC for NK92 and MDA-MB-231 by counting the number of calcein AM-positive cells over time
- The number of calcein AM-positive cells decreased as antibody dosage and time increased
- In addition, the ability to view bright field images allowed observation of the formation of immune complexes indicating cytotoxicity