Tran, Cuu Bao Chau Florence , Laliberte, Sylvie .
pHi changes in larch protoplasts over 33.5h of culture, as analysed with flow cytometry and using in vitro and in situ calibration of C-SNARF-1.
Many processes are regulated by intracellular pH and SNARF indicators are frequently used for pHi determination. Probes have been calibrated in vitro and in situ for confocal microscopy or spectrofluorometry and shown to behave differently once loaded into cells. This has generally been attributed to interactions with intracellular components generating over- or underestimations of pHi. We compared in situ and in vitro calibrations of C-SNARF-1 AM using flow cytometry. The first was performed with nigericin, while an in vitro calibration of the probe embedded in agarose pieces and de-esterified by porcine esterase was devised. Reproducibility was evaluated for both methods. The experimental system consisted of hybrid larch protoplasts, isolated from embryogenic suspensions and analysed at 2-3 days intervals over a 33.5h culture period. The experiment was repeated once and each sampling sequence involved two protoplast isolations. When compared, calibration curves showed pKa shifts and in situ curves differed more from each other than in vitro ones. pHi estimations during the first 21h showed a change from 6.26 to 6.89 when in situ calibration equation was used while values differed from 0.004 to 0.32 unit with in vitro equation. Following this alkalinization, pHi kept mostly over neutral value and fluctuated slightly; estimates from in situ and in vitro equations differed from 0.10 to 0.19 unit. As mentioned for other species, initial acidic values could reflect intracellular acidification caused by cell incubation in the digestion medium (pH 5.8) during protoplast preparation, while subsequent alkalinization might correspond to gradual pHi recovery towards homeostasis. The tendency towards a smaller amplitude in pHi changes observed after 23h of culture could be attributable to tighter pHi regulation. We cannot ascertain which, of in situ or in vitro calibration, generated better estimations of real pHi. However, the simultaneous use of both approaches allows some evaluation of the representativity of pHi values, through the range of variation between estimates from in situ and in vitro calibrations.
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1 - University of Quebec in Montreal, Biological sciences, C.P. 8888, Succ. Centre-Ville, Montreal, Quebec, H3C3P8, Canada
Presentation Type: Poster:Posters for Sections
Location: Khorassan Ballroom/Chase Park Plaza
Date: Monday, July 11th, 2011
Time: 5:30 PM