Technology - Screening Facility > Calibration Protocol

Purpose:  To calibrate the accuracy and precision of a 384 steel pin transfer device using a rhodamine solution in DMSO.

A. Prepare the Dilution Series for the Calibration Curve

• In labeled tubes prepare the following dilutions of rhodamine green (Rhodamine 110 Sigma/Aldrich #43,220-2) in DMSO from a 10mM stock solution.
• When preparing the dilution series, prepare enough of the 1.25uM stock (concentration E) to fill a 384-well Marsh plate with 40uL/well, a 384-well Genetix plate with 10uL/well one with 25uL/well, and an ABgene plate with 20uL (see Section C below, “Preparing the source plate for testing pin arrays”).

B. Make the Calibration Curve Plate:

• Because investigators usually aim for a 300-fold dilution of compound stocks into their assay plates, when doing calibrations, rhodamine DMSO stocks are diluted 300-fold into an aqueous buffer (PBS).
• Prepare Eppendorf tubes with 3ul of each concentration in 897ul of PBS
• Final concentrations for the curve are shown below.

  	Final Conc. (nM)
  A’	0.26
  B’	0.52
  C’	1.0
  D’	2.1
  E’	4.2
  F’	8.3
  G’	16.7
  H’	33.3

• With these prepared solutions, pipette the series into a 384-well black NUNC plate (Fisher #12-565-341) as diagrammed below.  Also add two full columns of 3ul of DMSO diluted in 897ul PBS as a background measure (X) (grayed boxes empty). 

Diagram of Control Plate for Robot Calibration  

C. Prepare Source Plate for Testing Pin Arrays 

• A final concentration of 4.2nM rhodamine green falls in the middle of the linear range for our Analyst plate reader.  Thus, this concentration was chosen as the goal for the final concentration in each well of the test target plates and the 1.25uM (E) rhodamine stock solution is used to fill the test source plates (384-well Marsh, Genetix or ABgene plates).  The volume for each well of the source plates is determined by the optimal volume for each plate type.

D. Calibration

• Rhodamine stock solutions from the source plates are then pin transferred into the test target plates (Black NUNC 384-well non-sterile; Fisher #12-565-341).  Several repetitions of transfer for each source plate into different target plates are carried out for each pin array being calibrated.
• The plates are then read on an Analyst plate reader with the following settings for Fluorescence Intensity measurements, with the fluorescein filter in place and the attenuator mode set to medium. 

E. Analyzing the Data

Currently a Macro being run from Excel is being used to calculate the final concentration of each calibration plate and volume of rhodamine transferred by each pin array.  The Macro calculates the data in the following way:

Making the Calibration Curve:

• The calibration curve plate should be the first one read on the plate reader and the resulting data is entered into a specific sheet in an Excel template.  This template enters each data set of the curve (six in all) into a table and subtracts the average of the background data before calculating the slope (m), and the y-intercept (b) of the curve.  

Reading and Analyzing the Results from Test Target Plates:

• The calibration curve (derived above) is used to calculate the amount of rhodamine actually transferred from the test source plates into the test target plates.
• The rhodamine concentration is calculated using the slope and y-intercept from the calibration curve and the known value of the E concentration from the curve and solving for x, the unknown concentration of the target well, from the equation for a line y = mx+b.
• The amount transferred per well is calculated by multiplying the amount in the assay well (in nanoliters) by the rhodamine concentration for that well, divided by the starting concentration of the rhodamine source plate in nanomoles (1250nM).
• In addition to calculating the amount of rhodamine transferred to each
  well, the standard deviation of transfer across each plate is calculated.
• Finally, the %CV for transfer into each plate is calculated by dividing
  the standard deviation of all the amounts transferred by the average of all
  the amounts transferred and then multiplying by 100. For pin transfer, a
  resulting percent CV of less than 15 is considered acceptable.