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

1st Reagent

2nd Reagent

3rd Reagent

4th Reagent

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