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The LSRFortessa is a digital cell analyser equipped with a Violet (405nm, 50mW), Blue (488nm, 50mW) and Red (640nm, 40mW) laser, capable of measuring up to 14 fluorescent parameters in addition to FSC and SSC. N.B. The warm up time for the instrument’s lasers = 30min. If you are the first user of the day, but are not based in the JR, please call ahead and we can switch on the instrument for you.

fortessa.jpg

The fluorochromes listed in the table below are examples of reagents which can be measured in each detector, but does not represent an exhaustive list. Please ask if you are unsure whether a particular reagent can be detected or not.

Violet Blue Red

A.       BV785 or BV786

B.       BV711

C.       BV650, QD655

D.       BV605, QD605

E.       BV510, HV500*

F.       BV421

A.       PE-Cy7

B.       PerCP, PE-Cy5.5

C.       PE-TxR, PE-CF694

D.       PE

E.       FITC, AF488, BB515

A.       APC-Cy7, APC-eF780, APC-H7

B.       AF700

C.       APC

 * The Fortessa can be set up to measure BV570 in this detector using a different software configuration (requires filter swap).

If you wish to measure any fluorescent parameters which are not listed above, please contact the Facility Manager  as we may have additional optical filters to fit your requirements. A more detailed instrument configuration including the dichroic mirrors and bandpass filters can be found below.

General information

  • Sample acquisition: >15,000 events/sec (2x107 cells/ml running at 60μl/min)
  • The ‘Sample fine adjustment knob’ allows you to adjust the sample flow rate to intermediate levels. When the knob is at its mid-point: LO = 12µl/min, MED = 35μl/min, HI = 60μl/min. Five full revolutions from the mid-point provide 0.5-2X the mid-point rate e.g. sample flow rates of 6-24µl/min are possible when the LO button is selected.
  • Allow at least 5 minutes for the pressure to stabilise after priming the instrument - this is crucial prior to running the CS&T as not doing so could result in erroneously calculated laser delays and suboptimal data.
  • Under no circumstance may the outer needle be removed when running samples.

LSRFortessa configuration

Laser PMT Dichroic mirror Bandpass filter Range, nm
Violet

A

B

C
D
E*
F

750LP

670LP

635LP
595LP
475LP
-

785/60

705/40

660/20
615/20
525/50
450/50

755-815

685-725
650-670
605-625
500-550
425-475

Blue

A

B

C

D

E

SSC

750LP

685LP

600LP

550LP

505LP

-

780/60

695/40

610/20

575/26

530/30

488/10

750-810

685-715

600-620

562-588

515-545

483-493

Red

A

B

C

750LP

710LP

-

780/60

730/45

670/14

750-810

710-750

663-677

* 575LP & 585/42BP used for BV570

(Image used courtesy of BD Biosciences)