Scintillation counter

L-[¹⁴C]-Cystine (PerkinElmer, 100 mCi/mmol) uptake was measured according to Giraudi et al. with some modifications^{59 (link)}. Cells were seeded in a 12-well plate pretreated with CCM and incubated for 6 h with HNE as previously described. Cells were then rinsed with warmed uptake buffer. Cystine uptake was started by incubating the cells in uptake buffer containing L-[¹⁴C]-Cystine 0.1 μCi at room temperature for 10 min, and stopped by rinsing with ice-cold unlabeled uptake buffer. Cells were then lysed by adding NaOH 0.1 N. Lysate was mixed with a scintillation cocktail (UltimaGold, PerkinElmer), protein quantification was done with the BCA protein Assay kit (Pierce, Rockford, II, USA) and radioactivity was determined using a scintillation counter (Hewlett Packard). Results were expressed as nmol of incorporated cystine reported to total protein content.

HepG2 cells (3 × 10³) were plated in 12 well tissue culture plates and allowed to adhere for 24 hr at 37 °C. Next day, cells were starved of serum and glucose for 2 hr. Thereafter, fresh DMEM containing 10% FBS with NG, HG, NG + Mntl and HG + CytoB was added and cells were cultured for 16 hr. Glucose uptake assay was performed as mentioned earlier50 (link). Radioactive glucose uptake was measured and represented as counts per million (CPM) in scintillation counter (Packard, MN, U.S.A.). For experiments in which time intervals are variable, HepG2 cells were plated as mentioned above and after serum and glucose starvation for 2 hr cells were cultured in NG or HG for 0, 3, 6, 12 or 24 hr. Thereafter, radioactive glucose uptake was measured as described above.

L. lactis was grown overnight in THB. A one ml sample was pelleted, washed three times in PBS, and used to inoculate minimal medium supplemented with either 1 nM or 1 μM of biotin. Samples were grown for 4.5 h, pelleted, and washed three times with PBS to remove external biotin. Pellets were then suspended in PBS plus 0.5% glucose for transport assays6 (link) and quantification of total protein in each sample using the Bio-Rad Protein assay. For transport assays, samples were incubated with 0.5 μM ³H-biotin at 30 °C for 0.5, 1, 2, 5, 30, or 60 minutes. Reactions were halted via dilution in ice cold PBS and bacteria were collected on 0.45 μm filters. Filters were then mixed with scintillation fluid and DPM were counted using a Packard Scintillation counter.

Prokaryotic cells were counted using a flow cytometer (FacsCalibur, Becton Dickinson, Heidelberg, Germany) following the method of Gasol et al. (1999) (link). Calculations were performed using the software program “Cell Quest Pro,” plotting the emission fluorescence of SYBR Green I (488 nm) vs. the side scatter. Picocyanobacteria were similarly counted on the basis of their signature in a plot of orange (FL2) vs. red (FL3) fluorescence.
The incorporation of ³H-leucine (140 Ci mmol^-1) was measured to estimate heterotrophic bacterial productivity in 10-mL water samples. Triplicate samples were incubated at a final concentration of 100 nM for at least 1 h at the in situ temperature in the dark. Incorporation was stopped by fixing the cells with formaldehyde (5% v/v). A fourth sample, serving as a blank, was fixed for at least 10 min prior to the addition of the radioactively labeled substrate. The samples were filtered onto 0.22-μm polycarbonate filters (Millipore), which were then placed in 4 mL of scintillation cocktail. The incorporated substrate was counted in a scintillation counter (Packard). Bacterial carbon production was calculated from ³H-leucine incorporation according to Simon and Azam (1987) (link), using a leucine mol% value of 7.3 and a carbon conversion factor of 0.86.

Immunophenotyping and mitogen-induced LP were performed to help characterize the structure and function of the dolphin's adaptive immune response. Immunophenotyping was used to differentiate specific types of immune cells and the proteins expressed by these cells in the adaptive immune response. Lymphocyte subsets were labeled and analyzed according to methods described previously (6 (link), 18 (link), 39 (link)–44 (link)). Lymphocytes were analyzed by a LSR flow cytometer (BD Biosciences, San Jose, CA, USA). Ten thousand lymphocyte-gated events were evaluated by histogram statistics (44 (link)).
LP is the first step in a functional adaptive immune response to create effector lymphocytes necessary for T cell and B cell mediated immune responses (37 ). The LP response was measured using techniques optimized previously (45 ). Briefly, isolated viable PBLs were incubated in well plates with concanavalin A (Con A; a T-cell mitogen), lipopolysaccharide (LPS; E. coli 055:B5; a B-cell mitogen), or supplemented RPMI-1640 representing unstimulated wells in triplicate followed by the addition of tritiated thymidine. Cells were then harvested and assessed using a scintillation counter (Packard, Meriden, CT, USA).

At initiation of treatment, [³H]-palmitic acid (1 μCi/ml medium) purchased from Perkin Elmer (32.0 Ci/mmol) was added to treated and untreated samples. At the indicated time points, lipids were extracted according to Bligh and Dyer method [49 (link)], N₂ dried, and resuspended in 60 μl chloroform:methanol (2:1); 40 μl were spotted on 20 cm silica gel TLC plates. Plates were developed with ethylacetate:isooctane:acetic acid (90:50:20, v/v), air dried, and sprayed lightly with En3hance® (Perkin Elmer) to enhance tritium readings. [³H]-Cer spots were visualized by iodine vapor mark. Radioactivity was visualized by autoradiography after 96 h at −80°C and the [³H]-Cer spots were scraped into scintillation vials containing 4 ml of scintillation fluid and counted on a Packard scintillation counter. [³H]-Cer counts were normalized to lipid phosphate levels.