SICKKIDS-UHN FLOW CYTOMETRY FACILITY
ASSISTED FLOW CYTOMETRY & DATA ANALYSIS FORM
Name
PI:
User:
Experiment Date:
Contact information
Location:
Email:
Phone #:
Instrument Selection & booking: is made by FCF staff based on your experiment and biosafety requirements
Acquisition time required: depends on # of samples and events recorded. Min of 30 minutes is required for instrument setup and
cleaning.
CELLS & CELL PREP INFORMATION
1. Species origin of cells (e.g. Human, mouse)
2. Tissue Source (e.g. spleen, lung)
3. Primary or cell line (if cell line provide name)
4. # of cells per sample
5. Estimated frequency of population of interest?
6. Buffer used to re-suspend cells?
7. Are these cells adherent?
8. Cell size (~um)
9. Are these cells prone to clumping?
10. # of events to acquire?
11. Was a viability stain used (if yes, please specify)
12. Are these cells expressing any fluorescent proteins (e.g. RFP,
GFP)
BIOSAFETY INFORMATION
All analysis must be performed in compliance with the SickKids-UHN TMDT Flow Cytometry Facility Biosafety Policy. For every
analysis appointment, clients must indicate the types of samples to be analyzed and identify the Risk Group (RG) categories of
any biological agents their samples contain, based on the Laboratory Biosafety Guidelines (3rd Edition, 2004, Public Health
Agency of Canada-PHAC, as enforced by the Human Pathogens and Toxins Act) and Containment Standards for Veterinary
Facilities (Canadian Food Inspection Agency-CFIA, 2007) Risk Group Classifications. A guideline for assessing RG categories of
your samples can be found at http://www.absa.org/riskgroups/.
The SickKids-UHN TMDT FCF operates in CL2 facilities using CL2 operations as a minimum standard of practice. Therefore,
FCF facilities are capable of handling samples designated as RG1 or RG2. No RG3 and RG4 agents are permitted.
Please consult TABLE 1: FCF Biosafety Requirements for Analysis and Sorting of Different Sample Types to determine whether
your samples need to be fixed prior to analysis.
13. Risk Group of samples (RG1,RG2,RG3 or RG4)
14. List any chemical treatments (e.g. PMA, Ionomycin) and
associated risk (e.g. carcinogen)
15. List any infectious biological agents cells have been
exposed to (e.g. bacteria, retrovirus, EBV)
16. Do cells/biological agents require Level 3 operating
procedure as determined by Sickkids/UHN Research
Biosafety Committee ? If yes, identify cells/biological
agent(s) and Risk Group
17. Have the cells been infected or transfected with any
gene transfer vector, and if yes, which one?
18. Are cells fixed? (Yes or No)
If yes, list fixative if it’s not the recommended (0.5-2% PFA
for at least 30’)
SAMPLES MUST BE FILTERED IMMEDIATELY PRIOR TO RUNNING (100uM size filter)
FCF can only use 5ml FACS tubes for acquisition.
(Corning Inc, cat # 352052, 5ml Polystyrene Round-Bottom tubes)
Experiment goal:
CONTROLS & EXPERIMENTAL SAMPLES
Example
CONTROL 1
CONTROL 2
CONTROL 3
CONTROL 4
CONTROL 5
CONTROL X
SAMPLE 1
SAMPLE 2
SAMPLE 3
SAMPLE 4
SAMPLE 5
SAMPLE 6
SAMPLE 7
SAMPLE 8
SAMPLE 9
SAMPLE 10
SAMPLE 11
SAMPLE 12
SAMPLE 13
SAMPLE X
NAME
Marker 1 +
Fluorochrome
Marker 2 +
Fluorochrome
Marker 3 +
Fluorochrome
Marker 4 +
Fluorochrome
Marker 5 +
Fluorochrome
Marker 6 +
Fluorochrome
Viability
wtSpleen
Untreated
pSTAT5 PE
pLCK Ax647
CD4 eF450
CD8a PE-Cy
TCRab PerCPCy5.5
B220 NC605
DAPI
UNSTAINED
x
x
x
x
x
x
x
EXPERIMENTAL CONTROLS INFORMATION
Instrument Setup and Gating Controls:

An UNSTAINED CONTROL tube (one for each tissue/cell line) is required for instrument set up.
*** YOU MUST BRING THIS CONTROL FOR ANY EXPERIMENT

All multicolour assays require SINGLE STAINED CONTROLS for each fluorochrome (cells and/or beads) to calculat e
compensation.

Viability dye alone control if using viability dye.

Fluorescence Minus One (FMO) controls for multicolour assays: used to identify and gate cells in the context of data spread due to
the multiple fluorochromes in a given panel. An FMO control contains all the flurochromes in a panel, except for the one that is being
measured.
Experimental Controls:

Dependent on experimental requirements and can include the following:

UNTREATED UNSTAINED, UNTREATED STAINED, TREATED UNSTAINED and TREATED STAINED samples.

Vector ALONE or UNINFECTED.

Positive/negative staining controls.

Staining specificity controls: secondary alone. knock-out, siRNA samples