Dear Maria
You ll probably have to lower the probenecid dose to maybe half or less, and play a little with its concentration. All you want is to prevent dye extrusion  at MINIMUM physiological cost of probenecid to the cells.

I suggest   Loading medium: Add an equal volume of 2X Fluo4 Direct reagent
without a quarter or fifth of the Probenecid you normally use  to cells containing culture medium (0%FCS) (1:1).

-         Incubate the cells for 30min at 37ºC

-         Remove cell medium for fresh medium without (I would actually add a percent or half of FCS to give a sharper set inhibition of  enzyme activity, give the sells some nutrient , and allow them to experience a    a small Ca reversible Ca boost to "wake them up" before you start officially measuring Ca ). For measurements you can remove FCS again.


 Rationale: you have to get rid of the fluo 4 to prevent compartimentalisation (medium exchange after 30 min loading time OK), to get    as little as possible fluo-4 as reactive as possible. Your experiment without probenecid was actually quite well.  And that at minimum probenecid necessary.

Alternatively, you could incubate a little longer with fluo 4 to raise a little its concentration (incubating 35 min), and add probenecid (fifth of your concentration used )  10 min after you started loading your cells, and maybe keep it during Ca recording.


I hope it helps

Thanks

Axel   Astar IMCB Central Imaging  , Singapore


-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Maria Calvo
Sent: Tuesday, March 02, 2010 11:58 PM
To: [log in to unmask]
Subject: Problems with Fluo4- Direct

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]
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