Sort Resources

Using the Sort Calculator# to determine optimal drop delay

Introduction

One of Rmax's properties is that it can accuratly calculate the drop delay. After using standard methods such as slide or bead systems to set the delay, you can use your sort sample itself to eveluate the optimal drop delay by scanning +/- 3 fractions of a drop delay. Graph the result and locate the peak delay and dial the value into the cytometer. This is your optimal delay point.

Materials

•  Beads with at least two populatons

–  E.g. BD Calibrite 3 pack (349502 BD Calibrite™ BD Calibrite Beads Unlabelled, FITC and PE Beads)

•  Centrifuge

•  Sonicator

•  Analysis tubes such as BD Falcon (Polystyrene BD2035)

•  2% to 5% BSA PBS stock solulon (100mls)

•  Cytometry toolkit sotware for Android and iOS. https://sites.google.com/site/cytometrytools/home/sotware

•  or the excel spreadsheet hosted on the ECN (European Cytometry Network) http://www.eurocyt.net

Preparaton

Using a BD 5ml tube:

•  Add 3 drops of Calibrite PE and FITC

•  Add 1ml of 5% BSA PBS and sonicate for 1 to 5 minutes

Instrument

•  Set the instrument up as usual

•  Set up a sort gate to take one populaton

•  Set the sort logical so that only one drop will be deflected and only targets

•  Start the sort and collect about 4mls of centre stream catch.

Centre stream catch (CSC) MoFio

Centre stream catch (CSC) Aria

(Picture by Rui Gardner IGC Portugal)

Method

•  Adjust the drop delay and recapture the centre stream

•  Do this unil you have +3 and -­3 delay steps of a drop around the instrument’s calculated delay

•  Centrifuge the beads for 2 mins at 300g

•  Decant off the supernatant leave 300μl

•  Acquire samples on a analyser (and the original) and analyse the target populaton

Data Analysis: Moflo Example

The original sample...

Draw the gate over the target population, then analyse all 7 CSC samples

•Tabulate the Total numbers and the Target numbers

Using the Rmax# Calculator

• Enter the Original sample Target and Total from the table and then just change the CSC fields for each tube run in the drop delay scan and record the Rmax%

• Graph the result and enter in the peak Rmax as the delay...

The Optimal Drop delay

• Enter the Optimal Drop Delay for your instrument and sort.