Flow Cytometry

Flow cytometry is a laser-based technology used to quantify specific properties of individual cells at a rapid rate. Prior to flow cytometric analysis, cells are prepared as a single-cell (monodispersed) suspension in a liquid buffer or medium. Depending upon the properties to be measured, the cells may be treated with one or more fluorescent reagent, whose binding to the cell is indicative of the cellular characteristic under investigation. During analysis, the cells are passed, single-file, past one or more lasers that are used to stimulate the fluorescence of reagents used. The intensity of fluorescence of each of the reagents used to treat the cells is detected and quantified by the flow cytometer for each cell that is detected. Usually, 10,000 cells are analyzed in less than a minute.

Cell properties that are typically analyzed by flow cytometry include, but certainly not limited to:

  • Relative size
  • Relative granularity
  • DNA content
  • RNA content
  • Expression of specific cell surface molecules
  • Expression of specific intracellular molecules
  • Expression of fluorescent proteins
  • Transient cell signaling events

In addition to the analytical flow cytometry described above, some of these instruments (cell sorters) are capable of physically separating selected subsets of cells from the original heterogeneous population for further growth or analysis.

The Flow Cytometry Lab of the Research Core Facility offers a very robust complement of flow cytometric services to investigators, including simultaneous analysis of up to 17 parameters and cell sorting of up to 4 cellular populations, again based on up to 17 parameters.

FAQs

How much sample volume do I need for flow cytometry? 

A minimum of 350 µL for analytic flow & 500 µL for sorting flow (in single cell suspension).

What should my cellular concentration be?

For analytic flow approximately 1 x 106 (a million per mL).
For sorting flow 8 -10 x 107 (8 – 10 million per mL).

What kind of controls do I need?

A bare minimum would include:

  1. Unstained
  2. An isotype antibody conjugated to your fluorochrome of choice (FITC, PE, APC etc.).
  3. If 2 or more colors used, single color controls will be necessary.

 See below for an excellent example 

Tube   

Cells

1stReagent

2ndReagent

3rdReagent

4thReagent

Comment

1

Type 1

-

-

-

-

Unstained Cells

2

Type 1

Iso-FITC

-

-

-

Isotype Control

3

Type 1

-

Iso-PE

-

-

Isotype Control

4

Type 1

-

-

Iso-PE/TR

-

Isotype Control

5

Type 1

-

-

-

Iso-APC

Isotype Control

6

Type 1

Ab1-FITC

-

-

-

Single Staining

7

Type 1

-

Ab2-PE

-

-

Single Staining

8

Type 1

-

-

Ab3-PE/TR

-

Single Staining

9

Type 1

-

-

-

Ab4-APC

Single Staining

10

Type 1

Iso-FITC

Ab2-PE

Ab3-PE/TR

Ab4-APC

Ab1 FMO Control

11

Type 1

Ab1-FITC

Iso-PE

Ab3-PE/TR

Ab4-APC

Ab2 FMO Control

12

Type 1

Ab1-FITC

Ab2-PE

Iso-PE/TR

Ab4-APC

Ab3 FMO Control

13

Type 1

Ab1-FITC

Ab2-PE

Ab3-PE/TR

Iso-APC

Ab4 FMO Control

14

Type 1

Ab1-FITC

Ab2-PE

Ab3-PE/TR

Ab4-APC

Test Sample

CONTACT US

 

Paula Polk, MS
Assistant Director, Research Core Facility
ppolk@lsuhsc.edu
(318) 675-4939

 

David Custis, MS
Research Specialist, Flow Cytometry
dcusti@lsuhsc.edu
(318) 675-4174