TITLE: Annexin V/PI- Assay: Flow Based Medium Throughput Assessment of Apoptotic Response to Two and Three Drug Combinations 
AUTHORS: Dennis Juarez, dfruman

[DESCRIPTION]
Basic principals and protocol for medium throughput cell viability screening in suspension cells using Annexin V/PI staining on the Attune NXT used in the Fruman lab.

[STEPS]
SECTION: Basic Principals and Planning Before Proceeding
4. Cells in culture can only tolerate small amounts of the vehicle in which a drug is dissolved in.

As such, you should minimize and standardize the amount of vehicle used in an experiment across all treatments.

SECTION: Basic Principals and Planning Before Proceeding
4.1. I cap all experiments at 0.1% DMSO in the final experimental condition (1:1000 dilution of DMSO in media).
 

SECTION: Basic Principals and Planning Before Proceeding
4.2. Single drug treatments are thus prepared at  working dilutions that are 1000x their final concentration with DMSO.
SECTION: Basic Principals and Planning Before Proceeding
5.1. In regards to preparing your working dilutions, stepwise dilution results in greater precision than direct dilution.
 


SECTION: Basic Principals and Planning Before Proceeding
5.2.     

SECTION: Basic Principals and Planning Before Proceeding
1. In vitro viability experiments should not be underestimated; especially, when carried out in a higher throughput fashion. 

With a structured an organized plan, a 96 well two drug combination response surface experiment can be plated in 30 minutes; whereas, a poorly thought out and planned experiment can eat away several hours of your day and can lead to non-meaningful results as prolonged experimental setup can affect your media quality and cell quality. 
SECTION: Basic Principals and Planning Before Proceeding
2. Several dilutions are required. To standardize the discussion, it's crucial to understand these key terms:

Stock Concentration: Refers to the concentration of the aliquots in long-term storage. Often stock concentrations are based on solubility of a compound or a practical concentration for aliquoting. As such, stock concentrations can be many times more concentrated than the final concentration.
 
Working Concentration: Refers to the concentration of any intermediate dilution of a drug in vehicle or media. As such, they are any concentration besides 1x and the stock concentration.

Final Concentration: Refers to the concentration of a drug which a cell is exposed to for the duration of the experiment. This is also known as 1x.
SECTION: Basic Principals and Planning Before Proceeding
4.3. Each agent of two drug treatments are prepared at working dilutions that are 2000x their final concentration.
SECTION: Basic Principals and Planning Before Proceeding
4.4. Each agent of three drug combination treatments are prepared at working dilutions that are 4000x their final concentration.
SECTION: Basic Principals and Planning Before Proceeding
5. Precision is key when carrying out these assays. Minimize variation by stepwise dilutions and using volumes that are easy to pipet and verify.
SECTION: Basic Principals and Planning Before Proceeding
3.   

SECTION: Basic Principals and Planning Before Proceeding
6. Plan your plate set up to include all single drug controls. To accomplish this, I recommend setting up a matrix.

    

SECTION: Prepare Dilutions and Plate Drugs
8. Prepare your DMSO-based working dilutions sterilely. From your stock solution, make your DMSO-based working dilutions per your experimental design in step 3. 

 

SECTION: Prepare Dilutions and Plate Drugs
9. Dilute your DMSO-based working dilutions into prewarmed media from step 7 as depicted in step 3. Only make as much as you need for each drug. 

Calculate this by multiplying the volume you will use per well by the number of wells that will get the specific drug dose. Make extra. 
SECTION: Prepare Dilutions and Plate Drugs
7. Prewarm your media.
SECTION: Prepare Dilutions and Plate Drugs
10. Think of the vehicle column and rows of your matrix as a drug and prepare an appropriate dilution per step 3 of DMSO in media. Usually you'll need more of this than the other drug doses.
SECTION: Prepare Dilutions and Plate Drugs
11. Using 8 channel reservoirs and a multichannel pipet, distribute your drugs into a 96 well flat bottom.
To minimize tip usage, you can reuse tips for multiple wells receiving the same drug.
In order to minimize well to well contamination with other drugs, deliver your first drug on the bottom of the plate. Your second drug should be delivered on the side wall of the plate. Your third drug will be delivered on the opposite side wall of the plate.
SECTION: Prepare Dilutions and Plate Drugs
12. When plating of all the drugs is complete, place the plate in the incubator. Plan to minimize this amount of time without the cells to avoid unnecessary evaporation.
SECTION: Prepare Dilutions and Plate Drugs
12.1. If you have a lot of cell lines that you will be plating, you can count your cells before starting your dilutions and drug plating. If you have a couple of cell lines, then preparing them next will not be too time consuming.
SECTION: Preparing and Plating Cells
13. Count your cells and either resuspend in fresh media or dilute cells to get them to either 0.5 million per mL or 1 million per mL if carrying out a triple combination.
SECTION: Preparing and Plating Cells
14. If doing a triple combination, don't forget to add 1:1000 DMSO to the cells.
SECTION: Preparing and Plating Cells
15. Transfer the appropriately concentrated cells to a reservoir and pull your plate with drugs from the incubator.
SECTION: Preparing and Plating Cells
16. Deliver cells by multichannel to an unused side wall.
SECTION: Preparing and Plating Cells
17. Incubate from 24-72 hours depending on your experiment.
SECTION: Annexin V PI Staining and Readout
18. After completion of incubation, transfer 100 uL of cells to a V-Bottom well plate. Centrifuge at 500g x 5 min
 




SECTION: Annexin V PI staining
18. 
SECTION: Annexin V PI Staining and Readout
20. After 5 minutes, the samples should be ready to read on the attune with the high throughput setting checked.
SECTION: Annexin V PI Staining and Readout
19. Resuspend in 100uL of 1:2000 Annexin V and 1:2000 propidium iodide diluted in annexin V binding buffer and transfer to a U-Bottom well plate.