Standard Operating Procedures for Sample Preparation for NCI60 Screen

General

NCI60 testing is performed in two parts: first a single concentration is tested in all 60 cell lines at a single dose of 10 ^-5 molar or 15 µg/ml. If the results obtained meet selection criteria, then the compound is tested again in all 60 cell lines in 5 x 10 fold dilutions with the top dose being 10-4 molar or 150 µg/ml. Compounds accepted for NCI60 testing are prepared for both 1-dose and 5-dose testing at the same time.

Agents Received

Synthetics & Pure Compounds with known molecular weight and macromolecules and compounds without molecular weights. Aliquots of agents identified for testing by DTP staff are weighed and transferred into pre-weighed glass vials, and labeled with a barcode, by staff at the Storage Contractor and transported to NCI–Frederick via courier service. Upon receipt, the contents of each package are checked against the accompanying shipping list. Compounds are not re-weighed, but are solubilized in these vials. Except when specifically noted, all agents are stored in a -70°C freezer.

Crude Natural Products. Crude Natural Product extracts are selected by the Natural Products Division staff and plated on detachable polypropylene (PP) 96-well microtiter plates. Plates are dried and stored at the Natural Products Repository @ -20 °C until called up for 1-dose testing. Based on the results of the 1-dose testing, those samples selected for 5-dose testing are rearranged from 88 wells to 72 wells per 96 well plate with a column of standard agent, Adriamycin, NSC 123127, and a new platemap is created and uploaded for assignment. Extracts are then solubilized at 40 mg/ml in DMSO or water.

Compound Requirements

Two factors influence the quantity of material required and used for each solubilization:

1. Screener Concentration Requirements
2. Screener Volume Requirements.

Concentration Requirements – 1-dose/Cancer in vitro program

Synthetic agents for the Cancer screen with a known molecular weight are prepared in DMSO:glycerol 9:1 (unless otherwise noted) at a concentration of 4 mM for the one dose assay and 40 mM for the 5-dose assay. In both cases the solution is diluted 1:400, giving a High Test concentration of 10 or 100 µM respectively. Synthetic agents (macromolecules) without a molecular weight are prepared in DMSO:glycerol 9:1 (unless otherwise noted) at a concentration of 6 and 60 mg/ml which is diluted 1:400, giving a High Test concentration of 15 and 150 µg/ml.

Natural products crude extracts which are organic solvent soluble are prepared in DMSO, while those which were produced by aqueous extraction are solubilized in water, both at 40 mg/ml.

Volume requirements – Prescreen/Cancer in vitro program

The Cancer screen requires 100 µl for 1 log, 5-dose dilutions of regular compounds and 75 µl for 1-dose testing. 1-dose testing is done at 1/10th the high concentration of 5-dose testing, so the volume requirement is 210 µl + 20% at 40 mM for compounds with molecular weights or 210 µl + 20% at 60,000 μg/ml = 250 µl for compounds without molecular weights (macromolecules) (i.e. less than 10 mg for MW = 1000 or 15 mg for compounds tested as weight/volume).

Compound Special Instructions

The special instructions, (e.g. oxygen sensitive, light sensitive) can change the handling of the agent according to the instructions.

Fresh Compounds

Compounds that are identified as needing to be prepared fresh before use are solubilized no more than one hour before serial dilution. It is serial diluted on a TECAN Freedom 200 (two drugs/plate), transferred to a column plate and stored under nitrogen in a desiccator box until delivered to the testing lab.

Solubilization Standard Operating Procedures

Entering Information into the NPSG TECAN System

Prior to beginning the solubilization procedure, information must be entered into the NPSG TECAN (Visual Basic instrument control and front end to ORACLE) system for each compound to be solubilized by the TECAN Freedom 200. A set of 72 compounds are assigned to a plateset by entering the shiplist numbers. A shiplist is loaded into TECAN software which looks up quantity and MW (from DIS Oracle tables) and calculates volume of solvent to be added to each vial to get constant concentration (40mM or 60,000 µg/ml) and adjusts concentrations if insufficient material for 1-dose and a test & one retest (75 µl @ 4 mM & 200 µl @ 40 mM + 20% or 75 µl @ 6,000 µg/ml & 200 µl @ 60,000 µg/ml + 20%). A Platemap (defines which compound is in which well) for prescreen is uploaded via ORADIS to ORACLE PLATEWELL table.

Supplies and Equipment

Vials are put on the TECAN table in shiplist order as designated on the PLATEMAP printout. TECAN Freedom robot adds appropriate volume of solvent (methanol/ethyl acetate/methyl-t-butyl ether, 6:3:1) to each vial to give constant concentration. Technician inspects each vial individually and sonicates, warms, etc. to achieve solution keeping the time of exposure less than two hours. Plate Preparation prior to drug solution transfer: Technician prepares three 96 well PP detachable well plates (one for 1-dose and two for 60 cell testing): 100 µl of 10% glycerol in isopropanol is added to each well. [After drug solution addition and vacuum drying, this leaves 10 ul glycerol per well.]

TECAN mixes drug solution in vial once then transfers 40 µL (400uM) of drug solution into each well of the 96 well detachable plate for 1-dose and 400 µL (4,000 µM) into 96 well PP detachable well plates for full 60 cell screen. All plates are transferred to the SpeedVac system for drying. All solvent is removed by high vacuum without heating leaving a residue of glycerol plus drug in bottom of the well. Dry plates are stored @-70 °C until called for test, typically 1-3 weeks. Untransferred residue in glass vials is dried in a SpeedVac and stored dry at -70 °C and can be used if additional retesting is required. If no retest is required, vials are incinerated after one year.

For 1-dose 60 cell testing: On the day of or the day before drug addition to growing cells in tissue culture, a strip of standards (adriamycin, NSC 123127 prepared and stored the same as the compounds) is added to the detachable well plate, and 90 µl DMSO is added to each well (4mM solution), and mixed/sonicated and 75 µl is transferred, using a 12 channel hand pipettor, to a 12 channel reservoir plates (column plates) , which is sealed and stored under nitrogen in a desiccator box until delivered to testing lab. The labels are placed at the right and the left of the front of the reservoir plate. It will be the first and the last NSC number in the row. Rows are transferred from detachable plate to columns 3-12 of column plates. Plates are sealed and stored under nitrogen no more than 24 hours prior to drug addition.

For 5-dose 60 cell testing: On the day of drug addition to growing cells in tissue culture, 90 µl DMSO is added to each well (40mM solution), and mixed/sonicated on the shaker of the TECAN Freedom 200 . Tubes are then placed on a TECAN Freedom 200 (two drugs/plate), and serial diluted/transferred to column plates which is sealed and stored under nitrogen in a desiccator box until delivered to testing lab. The plate labels are printed by the SATO thermal transfer printer utilizing the ORACLE front end program option AA- Expid NSC labels. The labels are placed at the right and the left of the front of the reservoir plate, drug one by column one and drug two by column twelve.

Vehicle Selection – Synthetics

The vehicles of choice are DMSO and water. Most agents are solubilized using one of these two vehicles. Other vehicles are used at the request of the supplier or based upon past testing methods. Agents utilizing volatile solvents as a vehicle are labeled 'Fresh' and are prepared within an hour of screening addition. Currently, all synthetic agents for Prescreen/Cancer screening are prepared in DMSO:glycerol 9:1, unless another vehicle is indicated. When water is indicated, the compound is solubilized in either distilled water or in cell culture media (RPMI 1640) without serum. All solubilizations requiring THF, Ethanol, Methanol, or other volatile solvents are prepared fresh to reduce evaporation.

Minimum Volume Requirements

The goal of solubilization is to deliver the highest requested concentration of an agent for the screening process. However, the number of vials required by the program screening the agent determines the minimum amount of vehicle that can be added. For the Cancer screening lab, if the amount of material sent is insufficient to create the required number of aliquots, the concentration must be dropped to ensure an appropriate volume is met. If it is a retest compound, permission must be obtained prior to dropping the concentration.

Volume requirements – 1-dose/Cancer in vitro program

The Cancer screen requires 100 µl for 1 log, 5-dose dilutions for the regular compounds. For compounds solubilized on the TECAN for both 1-dose and five-dose cancer assays, a minimum volume of 250 µl is needed, enough for the initial 1-dose assay, a test and a retest in the 5-dose assay.

Solubility Codes

The agents will not always solubilize to a clear solution absent of particles. Therefore the solution is described via a code best describing the solubility of the agent in the vehicle.

Special Note: It is the clarity of the solution that is being evaluated. Presence or absence of color is not accounted for in the solubility codes.

Labeling of Vials

One label must go on every vial that is prepared for freezing. This label must contain the Prefix, NSC number, shiplist number and plate and well number or concentration and volume or corrected amount. Labels are put on each vial so that the liquid inside each vial can be seen.One label must go on every vial that is prepared for freezing. This label must contain the Prefix, NSC number, shiplist number and plate and well number or concentration and volume or corrected amount. Labels are put on each vial so that the liquid inside each vial can be seen.