Hello everyone,

We are trying to optimize the  new Fluo4 direct (Invitrogen) for Cos-1 
cells and we have found different problems either for the cell loading 
and the response to ionomicyn (5 uM).

 Do you have any experience on this probe??  Could you tell me which 
loading protocol  you used? We have followed Invitrogen instructions.

Please find below more details of different protocols and results we got.

 Thank you, any help is appreciated,

1)

-         Loading medium: Add an equal volume of 2X Fluo4 Direct reagent 
with/ or without Probenecid to cells containing culture medium (0%FCS) 
(1:1).

-         Incubate the cells for 30min at 37ºC

-         Observate the cells under the microscope without removing the 
medium

PROBLEM: Cells were WELL charged with Fluo4 but DID NOT react to Ionomicin
 

2)        

-         Loading medium: Add an equal volume of 2X Fluo4 Direct reagent 
without Probenecid to cells containing culture medium (0%FCS) (1:1).

-         Incubate the cells for 30min at 37ºC

-         Remove cell medium for fresh medium without FCS

PROBLEM: Cells were NOT well charged with Fluo4 but DID react to Ionomicin

3)

-         Loading medium: Add an equal volume of 2X Fluo4 Direct reagent 
with Probenecid to cells containing culture medium (0%FCS) (1:1).

-         Incubate the cells for 30min at 37ºC

-         Remove loading medium for fresh medium without FCS

PROBLEM: Cells were WELL charged with Flou4 but DID react to Ionomicin 
very SLOW



Maria Calvo and Neus Abella

-- 


___________________________________

Dra. Maria Calvo

Unitat de Microscòpia Confocal
Serveis Cientificotècnics-C.Casanova
Facultat de Medicina
Universitat de Barcelona- IDIBAPS
C/ Casanova 143
Barcelona 08036

Tel: 34 934037159/39930
Fax: 34 934039946
E-mail: [log in to unmask]
___________________________________