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Pin Array Calibration Protocol

Purpose: To calibrate the accuracy and precision of a 384 or 96 steel pin transfer device using a rhodamine solution in DMSO.

A. Prepare dilution series for the calibration curve

  • Label and prepare tubes with the following dilutions of rhodamine green (Rhodamine 110 Sigma/Aldrich #43,220-2) in DMSO from a 10 mM stock solution.
  • When preparing the dilution series, prepare enough of the 1.25 uM stock (concentration E) to fill three ABgene 1056 plates with 10 uL (see Section C below, “Preparing the source plate for testing pin arrays”).
Label Stock Concentration
A 78 nM
B 156.5 nM
C 312.5 nM
D 625 nM
E* 1.25 uM
F 2.5 uM
G 5 uM
H 10 uM

B. Make the calibration curve plate

  • Since investigators typically aim for a 300-fold dilution of compound stocks into their assay plates, rhodamine DMSO stocks are diluted 300-fold into an aqueous buffer (PBS) during pin array calibration.
  • Prepare Eppendorf tubes with 3 uL of each concentration in 897 uL of PBS
  • Final concentrations for the curve are:
Label Final Concentration
A' 0.26 nM
B' 0.52 nM
C' 1.0 nM
D' 2.1 nM
E' 4.2 nM
F' 8.3 nM
G' 16.7 nM
H' 33.3 nM
  • Pipette 30 uL per well of these prepared solutions into a 384-well black Corning #3573 plate as diagrammed below.  To wells marked ‘X’, 30 uL diluted DMSO (3 uL into 897 uL PBS) is added to each well to quantitate the background value.  For low volume plates (eg: Corning #3821) three times the concentration of rhodamine green is used and 10 uL per well is pipetted to maintain dye concentrations in the linear range.

Note: Calibration curve plate is read in the EnVision plate reader at same time as the test target plates. Because rhodamine is light sensitive, keep this plate in light-protected drawer until ready to read with other plates.

C. Prepare source plate for testing pin arrays

  • A rhodamine green concentration of 4.2 nM was selected as the goal for the final concentration in each well of the test target plates and the 1.25 uM (E) rhodamine stock solution is used to fill the test source plate. The volume for each well of the source plate is determined by the optimal volume for each plate type.

D. Calibration

  • All test target plates to be used during calibration are filled with 30 uL/well PBS using the Combi (10 uL/well for low-volume plates and 150 uL/well for 96-well plates)
  • Rhodamine stock solutions from the source plates are pin transferred into the test target plates (black 384-well non-sterile plate, Corning #3573 for standard volume and Corning #3821 for low volume). 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 EnVision plate reader with the following settings for Fluorescence Intensity measurements, with the FITC filter set, (Ex: 485, Em: 535, DCM: 505)

E. Analyzing the data

An Excel Macro is utilized to calculate the final concentration of each calibration plate and volume of rhodamine transferred by each pin array.  The template calculates the data as follows:

Making the calibration curve:
  • The calibration curve plate is read first and the resulting data is entered into a specific region on an Excel template. Each data point of the curve (six total) is transferred into a table. The background average is subtracted then the slope (m), and y-intercept (b) of the curve are calculated. A minimum R2 value of 0.99 is accepted for calibration curve integrity.
Reading and analyzing the results from test target plates:
  • The calibration curve (derived above) is used to calculate the amount of rhodamine 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 (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 (1250 nM).
  • In addition to calculating the amount of rhodamine transferred to each well, the standard deviation of transfer across each plate is calculated.
  • The coefficient of variation (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. This value is multiplied by 100 to generate the % CV.  For pin transfer, a CV <10% is considered acceptable.