Assay Name: Antibody-Dependent Drug Uptake Assay

Transcription

1 Assay Name: Antibody-Dependent Drug Uptake Assay Assay ID: Celigo_02_0019 Table of Contents Experiment: Antibody-Dependent Drug Uptake Assay... 2 Celigo Setup...2 Assay Protocol and Plate Setup...3 Results Celigo gating function for fluorescent intensity analysis Celigo-captured Alexa Fluor 488 fluorescent images for suspension cells Percent cellular uptake of Alexa Fluor 488-drug for suspension cells Celigo-captured Alexa Fluor 488 fluorescent images for adherent cells Percent cellular uptake of Alexa Fluor 488-drug for adherent cells Conclusion Celigo Demonstration Experiment Antibody-dependent drug uptake assay 1

2 Experiment: Antibody-Dependent Drug Uptake Assay Purpose Current Method(s) Target Cell Type Experiment Plan Hypothesis Measure cellular drug uptake in adherent and suspension cell cultures of a fluorescently-labeled drug at varying antibody concentrations Flow Cytometry Human normal skin fibroblast Target cells are plated, then AF488-drug and antibody are added. Use the Celigo to scan the plate using bright field and green fluorescent channels The amount of drug uptake changes are dependent on antibody and drug concentrations Celigo Setup Plate Type Scan Channels Resolution Scan Area Analysis Method Scan Frequency Scan Time Greiner well black wall clear bottom Bright field and Green 1 µm/pixel Whole well Target 1 + Mask Once ~15 minutes Celigo Demonstration Experiment Antibody-dependent drug uptake assay 2

3 Assay Protocol and Plate Setup Goal The goal of this experiment is to measure cellular uptake of a fluorescently-labeled drug and varying antibody concentrations in an adherent and suspension cell culture. Protocol Suspension cell preparation 1. Skin fibroblasts were trypsinized and pipetted into a 96-well plate at 15,000 cells/well in 200 µl of media, following the plate map below 2. The cells were incubated overnight 3. After incubation, different concentrations of Alexa Fluor 488-Drug and antibodies were added to the wells and incubated for 3 hours (Plate Map Below) 4. After incubation, the cells were trypsinized and washed 5. The cells were reseeded at 200 µl in PBS into a new 96-well plate 6. The plate was imaged and analyzed using Celigo D2 D Ab20 A 15K 15K 15K 15K Ab15 B 15K 15K 15K 15K Ab10 C 15K 15K 15K 15K Ab5 D 15K 15K 15K 15K Ab2.5 E 15K 15K 15K 15K Ab1 F 15K 15K 15K 15K Ab0.5 G 15K 15K 15K 15K Ab 0 H 15K 15K 15K 15K Celigo Demonstration Experiment Antibody-dependent drug uptake assay 3

4 Adherent cell preparation 1. Skin fibroblasts were trypsinized and pipetted into a 96-well plate at 15,000 cells/well in 200 µl of media, following the plate map below 2. The cells were incubated overnight 3. After incubation, 2 different concentrations of Alexa Fluor 488-Drug and antibodies were added to the wells and incubated for 3 hours (Plate Map Below) 4. After incubation, the media was replaced with 200 µl of PBS 5. The plate was imaged and analyzed using Celigo Control D2 D1 D Ab20 A 15K 15K 15K 15K 15K 15K Ab15 B 15K 15K 15K 15K 15K 15K Ab10 C 15K 15K 15K 15K 15K 15K Ab5 D 15K 15K 15K 15K 15K 15K Ab2.5 E 15K 15K 15K 15K 15K 15K 15K 15K Ab1 F 15K 15K 15K 15K 15K 15K 15K 15K Ab0.5 G 15K 15K 15K 15K 15K 15K 15K 15K Ab0 H 15K 15K 15K 15K 15K 15K 15K 15K Data Collection 1. The plate was scanned in Celigo using Target for an endpoint reading a. Target 1 is the green fluorescent channel and Target 2 is the bright field channel Data Analysis The images for each drug and antibody concentration were analyzed to count total number cells in the bright field images Next, the fluorescent intensities were plotted in a histogram under the Gate Tab in the Celigo software Finally, the percentage of Alexa Fluor 488-positive cells were measured to determine the effect of antibody concentrations on drug uptake level Celigo Demonstration Experiment Antibody-dependent drug uptake assay 4

5 Results 1. Celigo gating function for fluorescent intensity analysis Cell images were analyzed by counting the total number of cells (Hoescht) and cells were gated based on the mean fluorescent intensity to determine percent of drug uptake Control, untreated (AF488+Hoescht) Drug 2, Ab 0.5 (AF488+Hoescht) Drug 2, Ab 20 (AF488+Hoescht) Celigo Demonstration Experiment Antibody-dependent drug uptake assay 5

6 2. Celigo-captured Alexa Fluor 488 fluorescent images for suspension cells Bright field and fluorescent images at different drug and antibody concentrations for suspension cells Representative images of cells incubated with Drug 2 and 1 at a high (20) and low (0.5) antibody concentration Celigo Demonstration Experiment Antibody-dependent drug uptake assay 6

7 % uptake of AF488-drug 3. Percent cellular uptake of Alexa Fluor 488-drug for suspension cells Total cells were counted using the bright field channel and the mean fluorescent intensities were measured within every counted cell Lower antibody concentrations incubated with the fluorescently-labeled drug resulted in higher percentage of uptake of the drug into the cells In the wells with higher antibody concentrations, drugs appeared to be aggregating extracellularly and were excluded from the fluorescent intensity data 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Antibody-Dependent Drug Uptake Assay Trypsinized Ab20 Ab15 Ab10 Ab5 Ab2.5 Ab1 Ab0.5 Ab0 Antibody concentration Drug 2 Drug 1 Celigo Demonstration Experiment Antibody-dependent drug uptake assay 7

8 4. Celigo-captured Alexa Fluor 488 fluorescent images for adherent cells Bright field and fluorescent images at at different Drug and Antibody concentrations for adherent cells Representative images of cells incubated with Drug 2, 1, and 0.5 at a high (20) and low (0.5) antibody concentration Celigo Demonstration Experiment Antibody-dependent drug uptake assay 8

9 Celigo Demonstration Experiment Antibody-dependent drug uptake assay 9

10 % uptake of AF488-drug 5. Percent cellular uptake of Alexa Fluor 488-drug for adherent cells Total cells were determined by Hoescht staining and bright field images were captured for morphological observation Mean Alexa Fluor 488 fluorescent intensities were measured from the area surrounding the nuclei for every cell counted 100% 90% 80% Antibody-Dependent Drug Uptake Assay Adherent 70% 60% 50% 40% 30% 20% 10% 0% Ab20 Ab15 Ab10 Ab5 Ab2.5 Ab1 Ab0.5 Ab0 Antibody concentration Drug 2 Drug 1 Drug 0.5 Conclusion Adherent cultures of human skin fibroblasts were prepared for a drug uptake assay Uptake of the fluorescent drug was inhibited at the higher antibody concentrations in both suspension and adherent cultures Using adherent cells in the microplate format allowed researchers to observe the morphological changes in the cell culture The results showed an increase in percent uptake as drug concentration increased In contrast, the percentage uptake increased as the antibody concentration decreased Scanning of one 96-well plate in three channels (bright field, green fluorescence, blue fluorescence) required less than 15 minutes Celigo Demonstration Experiment Antibody-dependent drug uptake assay 10