A241 CELL CULTURE

Problem 9 Count & Sort

Republic Polytechnic Singapore
Activity Owner : Jasmine Heng (Dr) Inputs : Ritu Bhalla (Dr) Approved By : Ventris Dsouza (Dr) Module Chair : Ritu Bhalla (Dr)

Copyright 2011, by Republic Polytechnic, Singapore

BD FACSCalibur

The Problem
You were asked how the simplest use of the FACS equipment can replace the use of haemocytometers in cell culture labs You were presented with a scenario of cell transfection experiments using GFP constructs and asked how screening and obtaining transfected cells can be done.

Copyright 2011, by Republic Polytechnic, Singapore

What do you recognise?

FACS (flow cytometer) and haemocytometer are equipment used for cell culture work FACS may have similar and perhaps better functions than haemocytometer, such that FACS can replace haemocytometer FACS can be used to screen and obtain transfected cells from cell transfection experiments There may be alternative way to obtain transfected cells without using FACS
Copyright 2011, by Republic Polytechnic, Singapore

The Approach

What is FACS? What is a haemocytometer? How to screen and obtain transfected cells using FACS? What is the working principle of FACS? What can FACS be used for? Applications? What are other methods to screen and obtain transfected cells? What is needed? What is the procedure?
Copyright 2011, by Republic Polytechnic, Singapore

What is FACS?
Fluorescence Activated Cell Sorter (aka flow cytometer)

Separates the cells in suspension on the basis of

Size

Granularity (Shape)

Fluorescence

Makes measurements on particles or cells as they flow in a fluid stream one by one through a sensing point

Key feature : Separate measurements are made on each particle within suspension in turn, and not just as average values for whole population of cells
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What is a haemocytometer?
Device for enumeration of cells (by manual counting under a microscope!)
Blood

cells Cell Lines

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What is the working principle of FACS?

FACS measures multiple cellular parameters, based on light scattering and fluorescence, to purify physically distinct subpopulations of cells
1.

Light Scatter

dependent on cell size and shape

2.

Fluorescence

dependent on absorption of light by cell and subsequent re-emission of this light at a different frequency
Copyright 2011, by Republic Polytechnic, Singapore

How does FACS work?

Fluidics Optics Electronics

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FACS profiles of cells are usually displayed as dot plots or histograms

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Using antibodies in FACS

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What can FACS be used for?

Capabilities of FACS :
Cell

Counting Cell Sorting (size, shape, fluorescence) Surface Marker Analyzing

Applications of FACS : Estimation of cellular parameters such as nucleic acid content, cell cycle, enzyme activity, calcium flux, membrane potential, and pH
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How does FACS count cells?

Set Flow Rate of FACS : x ml per min Obtain cell counts : y cells per min Convert to cell counts : y/x cells per ml
Note : The preparation and setting up of the FACS may not be practical for a single cell count, which is more conveniently done using a haemocytometer
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How to detect and obtain GFP-Transfected Cells using FACS?

Detection on a fluorescent microscope

Using FACS to analyze and sort cells without damaging them possible to culture specially selected cell populations
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How is FACS used to separate cells?

1. Grow transfected cells 2. Splits cells to get cells suspension 3. Prepare cells suspension for FACS 4. Run samples through FACS 5. Collect appropriate sorted fractions

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How to screen fluorescent cells without using FACS?

Trypsinise

Use Haemocytometer

fluorescence microscopy

cell count

Serially dilute down

Seed into the 1st row (96-well culture plate)

dilute

Get single cell in some wells at a particular dilution

Method of Limiting Dilution

Using fluorescent microscope, look for single fluorescent cell in the wells & grow cells from the single fluorescent cell (e.g. split confluent fluorescent cells in well and grow in flask)

Copyright 2011, by Republic Polytechnic, Singapore

You have learnt

FACS
principles Applications
Working

How FACS is used for cell counting How FACS is used for fluorescent cell sorting Other manual method for screening and obtaining transfected fluorescent cells eg. limiting dilution

End of Activity 9
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