European sea bass head-kidney leucocytes (HKLs) (n = 5 fish) were obtained and processed independently as previously described [42 (link)] and maintained in Leibovitz’s L-15 medium (Gibco, California, USA) supplemented with 10% foetal bovine serum (FBS), 2 mM glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 20 mM HEPES (Gibco). Later, HKLs were exposed through incubation of 107 HKLs/mL in 48-well microtiter plates (Nunc, New York, USA) at 22 °C during 24 h with: culture L-15 medium (control treatment), 5 μg/mL concanavalin A (ConA; Sigma-Aldrich, Darmstadt, Germany), 5 μg/mL lipopolysaccharide (LPS; Sigma-Aldrich), 10 μg/mL phytohemagglutinin (PHA; Sigma-Aldrich), 50 μg/mL synthetic unmethylated cytosine-phosphodiester-guanosine oligodeoxynucleotide 1668 (CpG ODN; sequence 5′-TCCATGACGTTCCTGATGCT-3′; Eurogentec, Seraing, Belgium), 25 μg/mL Poly I:C (pI:C; Sigma-Aldrich), 108/mL of Vibrio anguillarum (Va), or Photobacterium damselae (Pd) heat-killed bacteria and 106 TCID50 NNV/mL. After exposure, HKLs were washed with phosphate buffer saline (PBS) and conserved in TRIzol® Reagent at -80°C.
48 well microtiter plate
Manufactured by Thermo Fisher Scientific
Sourced in United States, Denmark, Germany, United Kingdom
The 48-well microtiter plates are a standard laboratory equipment used for various applications. They provide a platform with 48 individual wells, typically arranged in a 6x8 grid format, allowing for multiple samples or reactions to be processed simultaneously. The wells are designed to hold small volumes of liquids or suspensions, making them suitable for a range of assays, experiments, and high-throughput screening procedures.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (1 mentions)
Poly ic pic, by Merck Group (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Dm lb2, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
X vivo 10 media, by Lonza (1 mentions)
Human hemoglobin, by Merck Group (1 mentions)
Porcine hemin, by Merck Group (1 mentions)
Tryptic soy broth (tsb), by Thermo Fisher Scientific (1 mentions)
Equine myoglobin, by Merck Group (1 mentions)
6 protocols using 48 well microtiter plate
1
Immune response of sea bass head-kidney leucocytes
2
In Vitro Bacterial Adhesion Assay
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In vitro initial adhesion was assessed on PMMA and SS sur-faces (1 cm 2 slide per well) inserted in 48-well microtiter plates (Nunc, Denmark). A method developed by Simões et al. (2010) was used to allow the adhesion of single and dual species of B. cereus and P. fluorescens, for 2 h, on the selected sur-faces.
Total bacterial counts were obtained by direct staining with 4 , ,6-diamidino-2phenylindole (DAPI, Sigma, Portugal) as described previously (Saby et al., 1997) .
The slides were examined using an epifluorescence microscopy (LEICA DMLB2) as previously described (Lemos et al., 2013) .
Total bacterial counts were obtained by direct staining with 4 , ,6-diamidino-2phenylindole (DAPI, Sigma, Portugal) as described previously (Saby et al., 1997) .
The slides were examined using an epifluorescence microscopy (LEICA DMLB2) as previously described (Lemos et al., 2013) .
3
CD3-positive Cell Proliferation Assay
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Cell proliferation assays were carried out with Ficoll purified PBMCs as well as CD3-positive cells obtained using the different isolation methods. CD3-positive cells (5 × 105/500 µL) were cultured in X-VIVO 10™ media (Lonza Group Ltd., Basel, Switzerland) supplemented with 2% AB serum (Blood Service, Institute of Transfusion Medicine, University Hospital Leipzig, Leipzig, Germany), 100 U/mL IL-2 (PeproTech, Hamburg, Germany), in 48-well microtiter plates (Thermo Fisher Scientific, Waltham, MA, USA). The physiological activation of CD3-positive cells was achieved by the addition of the Dynabeads™ Human T-Activator CD3/CD28 (anti CD3/CD28, Thermo Fisher Scientific, Waltham, MA, USA) at a bead-to-cell ratio of 3:1. Thereafter, the cells were incubated for 6 days at 37 °C in a humidified atmosphere with 5% CO2.
4
Rearing Honey Bee Larvae in Controlled Conditions
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Frames with open brood were collected from strong, queen right Apis mellifera scutellata colonies maintained at the University of Pretoria apiary (Pretoria, South Africa) during late summer (January -April 2013). Two day old worker honey bee larvae were grafted onto larval food in 48-well microtiter plates (Thermo Fisher Scientific, Rochester, New York), using the protocol from Aupinel et al. (2005) and Crailsheim et al. (2013) . The grafted larvae (Fig. 1) were kept in an incubator (Humidity chamber HCP108, Memmert GmbH & Co. KG; Germany) at 34 ± 1 °C and 95% relative humidity in darkness, simulating conditions within the hive. They were removed from the incubator once a day for feeding. Larvae were randomly assigned to plates and the plates were randomly assigned to the experimental or control groups. Each group contained at least 60 larvae. Mortality was recorded on a daily basis. Survival and body mass data were collected from nine colonies in total. Kaplan-Meier and Log Rank (Mantel-Cox) survival analyses were performed to test for differences in survival between the control and nicotine diets. Survival were reported as percentage overall survival. Body mass was compared between the control and nicotine diets using One-way ANOVA. The alpha level was set to 0.05 for all analyses and the data were tested for normality.
5
Bacterial Growth Kinetics Assay
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Bacteria were grown overnight in TSB. Cells were harvested by centrifugation, washed with PBS (for growth in RPMI/TSB) or RPMI with 10 μM EDDHA (for hb/heme dependent growth), adjusted to an OD600 = 1 and 2,5 μl were used to inoculate 0,5 ml of the appropriate test medium in individual wells of a 48 well microtiter plate (NUNC). Bacterial growth was monitored for 24–48 h using an Epoch2 reader (300 rpm shaking at 37°C). The OD600nm was determined every 15 minutes. Bacterial generation times during exponential phase were calculated using the following formula (G- generation time, t0- start of exponential phase, t1- end of exponential phase, b- OD600t1 , B-OD600t0 .
6
Bacterial Growth in Iron-Limited Conditions
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All bacterial strains generated and/or used in this study are listed in Key resources table. For growth in iron limited conditions, bacteria were grown overnight in Tryptic Soy Broth (TSB) (Oxoid). Cells were harvested by centrifugation, washed with RPMI containing 10 µM EDDHA (LGC standards), adjusted to an OD600 = 1 and 2,5 µl were used to inoculate 0,5 ml of RPMI+ 1% casamino acids (BACTO) + 10 µM EDDHA in individual wells of a 48 well microtiter plate (NUNC). As sole iron source 200 nM porcine hemin (Sigma), 2.5 µg/ml human hemoglobin (own preparation), 10 µg/ml human myoglobin (Sigma) or equine myoglobin (Sigma), 117 nM human haptoglobin-hemoglobin or 200 nM hemopexin-heme (Sigma) were added to the wells. Bacterial growth was monitored using an Epoch2 reader (300 rpm, 37°C). The OD600 was measured every 15 min.
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