Aug 30, 2022
SPARC - Attune NxT Set-up for Milli-metabolic bead assay Acquisition
- 1Purdue University
Protocol Citation: J Paul Robinson 2022. SPARC - Attune NxT Set-up for Milli-metabolic bead assay Acquisition. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvro3o3vmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 19, 2019
Last Modified: August 30, 2022
Protocol Integer ID: 31158
Keywords: flow cytometry method, fluorescence detection, bead assay, hormone assay
Abstract
This protocol is to demonstrate how to run a standard plate-based assay on the flow cytometer to produced appropiate concentations of the various analytes to be measured.
Guidelines
This assays is specifally designed for the ATTUNE NxT flow cytometer and the Milli-metabolic bead assays
Materials
MATERIALS
Attune Performance Tracking BeadsThermo ScientificCatalog #4449754
Attune Focusing Fluid 1x10LThermo ScientificCatalog #A2490
Attune Wash SolutionThermo ScientificCatalog #A24974
Atttune 1x Shutdown SolutionThermo ScientificCatalog #A24775
12x75mm test tubeSarstedtCatalog #55.476.300
Before start
Setting up instruments correctly is a critical step in any assay
Check the waste and
If the waste is full, disconnect the tank and dispose of the waste down the sink with running water. Add 200 mL of 5% bleach to the tank and reconnect to the Attune NxT. Fill the Focusing Fluid tank from the focusing fluid cube.
Check and fill as needed the shutdown solution reservoir and the wash solution reservoir.
Also check the waste and focusing fluid reservoirs at the plate sampler station.
At the workstation, click on instrument > start-up at the top of the screen.
When start-up is complete, the indicator lights at the instrument will be green.
Run the performance test.
When the plate finishes, right click on the experiment.
Select export FCS files
Copy files onto the appropriate directory
Analyze data in Mplex software
Once data files are analyzed in MPLEX, follow the MPLEX protocol to send data for depositing to NIH. See this protocol for details:
Protocol
NAME
SPARC - Analysis of multiplexed bead data using MPLEX softwareCREATED BY
J Paul Robinson