NK92 cell-mediated ADCC using calcein AM

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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 Β΅m/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

  1. MDA-MB-231 were obtained from ATCC and cultured in RPMI 1640 media
  2. NK92-CD16 cells were purchased and expanded in RPMI 1640 media
  3. MDA-MB-231 cells were stained with 10 Β΅M of calcein AM (Nexcelom, Cat# CS1-0119) for 30 min and then washed 3 times with RPMI media
  4. The cells were then seeded into the 96-well plate at 10,000 cells/well
  5. 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 Β΅g/ml; and control antibody
    1. The spontaneous release samples were stained Target cells without Effector cells

plate map 96-well

Data Collection

  1. After adding the cells and antibodies, the plate was centrifuged to settle the cells to the bottom
  2. Immediately after, the plate was scanned in Celigo using Target 1 (Green) + 2 (BF) for t = 0 h
  3. 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

  1. The calcein AM-positive Target cells co-cultured with and without Effector cells were counted and recorded
  2. The calcein AM-positive Target cells with and without antibodies were counted and recorded
  3. The calcein AM-positive Target cells only were counted and recorded
  4. Normalized to t = 0
    cytotoxicity percentage normalized to time
  5. Normalized to spontaneous release
    % πΆπ‘¦π‘‘π‘œπ‘‘π‘œπ‘₯𝑖𝑐𝑖𝑑𝑦 = % π‘π‘¦π‘‘π‘œπ‘‘π‘œπ‘₯𝑖𝑐𝑖𝑑𝑦 (π‘ π‘Žπ‘šπ‘π‘™π‘’) βˆ’ % π‘π‘¦π‘‘π‘œπ‘‘π‘œπ‘₯𝑖𝑐𝑖𝑑𝑦 (π‘ π‘π‘œπ‘›π‘‘)
  6. 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

Celigo plate view results

  • 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

MDA-MB-231 different time points

  • Dose-dependent cytotoxicity results at different time points

dose dependent cytotoxicity results

  • 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

formation of immune complex clusters

  • 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

immune complex clusters 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
2018-10-11T18:09:28+00:00