Protocols and Guidelines

PFCC Guidelines - Getting started in:

Flow Cytometry Analysis

(1) Read our Guidelines A to help prepare for your flow experiment.

(2) Getting Started with Full Spectrum Flow (coming soon)

Cell Sorting

(1) Read our Guidelines S to help prepare for your sorting experiment.

• Basic FACS Buffer recipe and others
• Basic recommendations for Collection Buffer recipes (coming soon)

(2) Use our helpful Cell Sorting Calculators to decide what is the appropriate time to book for your experiments.

(3)  Do your final check using the Cell Sorting Checklist

Mass Cytometry

[EXT] Mass Cytometry/CytOF (Helios) Resources by UChicago

Small Particle Analysis

PFCC Guidelines NS for use of NanoSight NS300

PFCC Guidelines for analysis on Aurora and NIST Calibration files: (coming soon)

• PFCC Aurora "Abby Cadabby"
• PFCC Aurora ESP "Pascal"
• CIL Aurora ESP "Frida"

OTHER

PFCC Operator Assist Form

other coming: Recommended Sorting supplies, Guidelines for plate sorting. Do you have any requests? Submit your suggestion here: SUGGESTION BOX!

Protocols and Recommendations

Cell Harvesting and Preparation

Worthington Tissue Dissociation Guide: Link to website

PFCC Mouse Spleen dissociation protocol

more coming soon

Flow Cytometry Staining

OMIPs: check this published Optimized Multicolor Immunofluorescence Panels for flow cytometry. These can provide you a starting point and alleviate the development time in need of the same or highly similar panels.

Current Protocols in Immunology: research published protocols in this journal, and other Current Protocols multidisciplinary collections.

[EXT] BioLegend Technical Protocols: check here several standard staining protocols, aided with video tutorials.

more coming soon

Mass Cytometry Staining

Bio-Protocol 2015: "Intracellular Cytokine Staining on PBMCs Using CyTOF Mass Cytometry"

Bio-Protocol 2015: "Cytokine-stimulated Phosphoflow of PBMC Using CyTOF Mass Cytometry"

Bio-Protocol 2015: "Cytokine-Stimulated Phosphoflow of Whole Blood Using CyTOF Mass Cytometry"

Common Applications of Flow Cytometry

RNA isolation from sorted cells - This paper has a detailed protocol for how to properly prepare a sorter for RNA isolation and sort cells with Trizol, RLT, and RNase inhibitor. It also includes an estimated yield of RNA from certain cell types.

Measurement and Characterization of Apoptosis by Flow Cytometry: [EXT] Reference [1] and [2]

PhosphoFlow

• Cytobank's Compatibility Buffer and Protocol Selection Tool
• [EXT] Garry Nolan's Current Protocol's publication
• [EXT] Jonathan Irish's PhosphoFlow Course

[EXT] 10x Genomics Guidelines for Sorting

[EXT] NanoString University