Hi Christian, have you considered the timing of the Ca flux, i.e. how long after addition of a given stimulus does the flux start and persist? The Array is nice but loading a plate and reading multiple wells takes some time - if you've induced Ca flux outside the device, it will take some time for the first sample to get to the cuvette, even if you plunk down the plate on the carrier immediately after pipetting. Worst of all, the next few wells will be read at varying intervals starting from plate insertion, which you'd have to take into account as well. Just my 2 cents, Guy Next generation therapeutic <http://www.ablynx.com/> antibodies Guy Hermans, PhD Project Leader Ablynx NV Technologiepark 4 B-9052 Zwijnaarde Belgium guy.hermans@ablynx.com tel: fax: mobile: +32 (0)9 261 06 57 +32 (0)9 261 06 27 +32 (0)486 788 551 <https://www.plaxo.com/add_me?u=30065269879&v0=994426&k0=2009290972> Add me to your address book... <http://www.plaxo.com/signature> Want a signature like this? -----Original Message----- From: Christian Gray [mailto:christian.gray@cbio.com.au] Sent: Monday, September 05, 2005 7:24 AM To: cyto-inbox Subject: Calcium Flux on a FACSarray (Advice please) I am interested in measure calcium flux in PBMC on a BD FACSarray. Although this machine is good in that it takes a 96 well plate, it has a limited range of lasers (532, 635) and filters (Yellow 585/42, Far Red 685 LP, Red 661/16 and NIR 780/60). No FITC!! I have found a calcium flux reagent from Molecular Probes called Calcium Orange (Ex/Em 549/5760. Has anyone used this in flow cytometry, and what method do you use? I have only found reference to its use in confocal. This reagent is florescent prior to ester cleavage, which makes it difficult to differentiate between uncleaved ester and Ca2+ bound. We do have the option of going elsewhere and using Indo-1 on a flow cytometer with a UV laser, are we wasting our time with Calcium Orange. Christian. CBio Limited Queensland, Australia. www.cbio.com.au
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