Optimization of the cryopreservation of biological resources,Toxoplasma gondii tachyzoites,using flow cytometry

The conservation of Toxoplasma gondii strains isolated from humans and animals is essential for conducting studies on Toxoplasma. Conservation is the main function of the French Biological Toxoplasma Resource Centre (BRC Toxoplasma, France, http://www.toxocrb.com/). In this study, we have determined the suitability of a standard cryopreservation methodology for different Toxoplasma strains using the viability of tachyzoites assayed by flow cytometry with dual fluorescent labelling (calcein acetoxymethyl ester and propidium iodide) of tachyzoites. This method provides a comparative quantitative assessment of viability after thawing. The results helped to define and refine quality criteria before tachyzoite cryopreservation and optimization of the cryopreservation parameters. The optimized cryopreservation method uses a volume of 1.0 mL containing 8 × 10⁶ tachyzoites, in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% foetal calf serum (FCS). The cryoprotectant additive is 10% v/v Me₂SO without incubation. A cooling rate of ∼1 °C/min to −80 °C followed, after 48 h, by storage in liquid nitrogen. Thawing was performed using a 37 °C water bath that produced a warming rate of ∼100 °C/min, and samples were then diluted 1:5 in IMDM with 5% FCS, and centrifuged and resuspended for viability assessment.     

Dead cell counts during serum cultivation are underestimated by the fluorescent live/dead assay

The live/dead fluorescent assay provides a quick method for assessing the proportion of live and dead cells in cell culture systems or tissues and is widely used. Dead cells are detected by the fluorescence produced when propidium iodide (PI) binds to DNA; PI and similar molecules are excluded from live cells but can penetrate dead cells because of their loss of membrane integrity. Here we investigated the effect of serum in the culture medium on the reliability of the method. We assessed viability of chondrocytes with/without serum using both a live/dead assay kit and also trypan blue staining. We found that after 2 days of culture, the DNA-binding dye PI could no longer detect dead cells if serum was present but they were readily detected in serum-free medium or if an inhibitor to DNase I was added to the serum-containing medium. Dead cells could be detected by trypan blue staining in all cultures. Hence dead cells are no longer detected as the DNase I present in serum degrades their DNA. DNA-binding dyes may thus not give a reliable estimate of the number of dead cells in systems that have been cultured in the presence of serum for several days.     

Optical Imaging of Hippocampal Neurons with a Chloride-Sensitive Dye: Early Effects of In Vitro Ischemia

Abstract: We determined if changes in intraneuronal Cl^? occur early after ischemia in the hippocampal slice. Slices from juvenile rats (14–19 days old) were loaded with the cell-permeant form of 6-methoxy-N-ethylquinolinium chloride (MEQ), a Cl^?-sensitive fluorescent dye. Real-time changes in intracellular chloride concentration ([Cl^?]_i) were measured with UV laser scanning confocal microscopy in multiple neurons within each slice. In vitro ischemia (26–28°C, 10 min) was confirmed by the loss of synaptic transmission (evoked field excitatory postsynaptic potentials) from pyramidal cells in area CA1. After ischemia and reoxygenation (10 min), MEQ fluorescence decreased significantly in CA1 pyramidal cells and interneurons. The decreased fluorescence corresponded to an ischemia-induced increase in [Cl^?]_i of ～10 mM. Pretreatment with the GABA_A-gated Cl^? channel antagonist picrotoxin (100 µM) blocked the ischemia-induced change in [Cl^?]_i. Analysis of the superfusates indicated that ischemia also caused a transient amino acid (GABA, glutamate, and aspartate) release that was maximal at ～10 min, returning to baseline shortly thereafter. Recovery from ischemia was confirmed by the return of synaptic transmission in area CA1, the return toward baseline of the ischemia-induced decrease in MEQ fluorescence, and exclusion of propidium iodide from MEQ fluorescent cells. Furthermore, pyramidal cells did not undergo cell swelling during this early phase of reoxygenation, as indicated by the volume-sensitive dye calcein. Thus, mild ischemia induces the accumulation of [Cl^?]_i secondary to GABA_A receptor activation, in the absence of cellular swelling or death. In contrast, depolarization of the slice with K⁺ (50 mM) decreased MEQ fluorescence significantly but caused cell swelling. Picrotoxin did not prevent the K⁺-induced increase in [Cl^?]_i. It is possible that an increased [Cl^?]_i, following either an ischemic event or an episode of depolarization, would reduce the Cl^? driving force and thereby limit synaptic transmission by GABA. To support this hypothesis, ischemia caused a reduction in the ability of the GABA agonist muscimol to increase [Cl^?]_i after 20-min reoxygenation.     

Calcein-AM is a detector of intracellular oxidative activity

Calcein-acetoxymethylester (calcein-AM) is a non-fluorescent, cell permeant compound, which is converted by intracellular esterases into calcein, an anionic fluorescent form. It is used in microscopy and fluorometry and provides both morphological and functional information of viable cells. In this study we have tested the response of calcein-AM to oxidation. In cell-free fluorometric assays, H₂O₂ and xanthine–xanthine oxidase induced a dose-dependent emission of the AM form but had no effects on calcein. Fluorometric and confocal microscopy tests on human fibroblasts confirmed that the cell permeant AM form is the actual sensor since its removal from culture medium, and its consequent back-diffusion, made the system insensitive to oxidative stimuli. In time-lapse confocal microscopy, calcein-AM detected changes in the intracellular redox state following direct oxidation (H₂O₂, xanthine–xanthine oxidase) and phorbol ester treatment. Comparative tests showed that calcein-AM sensitivity to oxidation is about one order of magnitude higher than other fluorescein derivatives. The absence of leakage, due to the presence of the probe in the extracellular compartment, and its low toxicity allow to perform experiments for prolonged times following the response to the same or different stimuli repeatedly applied. We propose calcein-AM as a sensitive tool for intracellular ROS generation in living cells with useful applications for real-time imaging in confocal microscopy.     

Regulation of volume-sensitive Cl- channels in multi-drug resistant MCF7 cells

The P-glycoprotein (P-gp) is thought to be involved in the regulation of volume-sensitive chloride channels. In this study, the possible coupling between P-gp and swelling-activated chloride channels has been examined in MCF7 cells with sensitive (MDR-), resistant (MDR+), and reversed resistant (MDR(REV)) phenotypes. Western blot analysis showed that incubation of cells with doxorubicin induced P-gp expression in a reversible manner. Exposure of MDR+ cells to hypotonicity resulted in an inhibition of P-gp activity while hypotonic challenges induced swelling-activated chloride currents (I(Cl-swell)) in MDR-, MDR+, and MDR(REV) MCF7 cells. While verapamil inhibited I(Cl-swell) in all cell types, doxorubicin and vincristine rapidly and reversibly inhibited I(Cl-swell) uniquely in MDR+. Intracellular dialysis of MDR+ cells with C219 anti-P-gp antibody abolished the sensitivity of I(Cl-swell) to doxorubicin and led to a response pattern very close to that of MDR- cells. Taken together, these results strongly suggest that the P-glycoprotein regulates I(Cl-swell) in resistant MCF7.     

Neutrophils are cytotoxic and growth-inhibiting for neuroblastoma cells with an anti-GD2 antibody but, without cytotoxicity, can be growth-stimulating

Neutrophils and mononuclear cells (MNC) can mediate antibody-dependent cellular cytotoxicity (ADCC) against cancer cells. To study cytotoxicity and growth inhibition of neuroblastoma cells by neutrophils and MNC with chimeric anti-disialoganglioside (GD2) monoclonal antibody (mAb) ch14.18, we developed digital image microscopy scanning (DIMSCAN) assays that measure fluorescence of target cells in 96-well plates after 6–18 h (cytotoxicity assay) or 7 days (growth assay). Neuroblastoma cell lines (GD2-positive: SMS-KCN, SMS-LHN, LA-N-1; GD2-negative: SK-N-SH) were preloaded with calcein acetoxymethyl ester for the cytotoxicity assay or labeled in situ after 7 days of culture with fluorescein diacetate in the growth assay. Fluorescence, as quantified by DIMSCAN, was correlated with neuroblastoma cell number in both assays (100–2000 cells/well). In the cytotoxicity test, both neutrophils and MNC effectively mediated ADCC of GD2-positive but not GD2-negative neuroblastoma cell lines. Cytotoxicity of both neutrophils and MNC increased with effector to target cell (E:T) ratio (5–50:1) and mAb ch.14.18 dose (0.1–10 μg/ml). ADCC of neutrophils, but not MNC, increased with addition of GM-CSF. Neutrophils, especially with rhGM-CSF, significantly suppressed growth of GD2-positive cell lines at a high E:T ratio (50:1) and mAb dose (10 μg/ml). Without antibody, neutrophils inhibited growth of one cell line (LA-N-1) but stimulated growth of two others (SMS-KCN, SMS-LHN). If neuroblastoma cells did not express GD2 (SK-N-SH), neutrophils stimulated growth whether or not antibody was present. Neutrophil culture supernatants increased growth of SK-N-SH, LA-N-1, and SMS-KCN cells, and MNC culture supernatants increased growth of SK-N-SH. In conclusion, neutrophils can mediate cytotoxicity and growth inhibition with a chimeric anti-GD2 antibody but also can promote tumor cell growth if antibody is not present or if GD2 is not expressed. Received: 18 November 1998 / Accepted: 24 September 1999