For detection of anti-Leptospira antibodies, microscopic agglutination test (MAT) was conducted according to the recommendations for international standards [22 ]. The complete panel included 28 serovars representing all the 24 known serogroups (from Royal Tropical Institute - KIT, Amsterdam, Netherlands). Bacteria were cultured in liquid Ellinghausen-McCullough-Johnson-Harris (EMJH) and used as antigens. Each serum sample was initially diluted 1:50 in buffered saline and 50 uL of this solution were transferred into vinyl microplates containing 96 wells (Corning Incorporated, Corning, NY, USA). Immediately after, an equal volume of each antigen was added to the corresponding well, with a final dilution of 1:100. The microplates were incubated at 37°C for 90 minutes and observed under dark-field microscopy. Serum samples were initially analyzed at a dilution of 1:100, and those that showed agglutination level equal to or higher than 50% were then diluted again in a geometrical ratio of two (1:200, 1:400 and 1:800). Samples were considered as reactive when reached titers of 200 or 400. Reactions that presented titres ≥800 were considered as strongly reactive and considered as an indication of acute infection [22 ].
96 wells
Manufactured by Corning
Sourced in United States
The 96 wells product is a laboratory equipment designed to hold and process multiple small-volume samples simultaneously. It features a grid of 96 individual wells, allowing for efficient and organized sample handling. The core function of this product is to provide a standardized platform for various laboratory applications that require the use of multiple samples in a controlled and reproducible manner.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
60 mm dish, by Corning (1 mentions)
Arpe 19 cell, by American Type Culture Collection (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Onalespib, by Selleck Chemicals (1 mentions)
Doxycycline, by Thermo Fisher Scientific (1 mentions)
40 μm nylon cell strainer, by Corning (1 mentions)
P nitrophenyl phosphate pnpp, by Merck Group (1 mentions)
10 protocols using 96 wells
1
Leptospirosis Antibody Detection by MAT
2
ARPE-19 Cell Culture Protocol
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A total of 2.5 × 106 ARPE-19 cells (ATCC, Manassas, VA) were seeded in a 60-mm dish (Corning, Tweksbury, MA) and cultured with Dulbecco’s Modified Engle’s Medium/F12 (Thermo Fisher, Waltham, MA) plus 10% of bovine serum albumin (Thermo Fisher) as well as 1% penicillin/streptomycin (Thermo Fisher) in a 37 °C humidified and 5% CO2 culture incubator. The cells were maintained in the 6 wells or harvested with trypsin (Thermo Fisher) and then seeded in 96 wells (Corning) before specific experiments.
3
Determining Total Phenol Content by Folin-Ciocalteu
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The total phenol content (TPC) was determined by Folin–Ciocalteu assay which referred to the method of Zhao et al. [16 (link)]. In total, 50 μL extracted sample was added to 3 mL DI Water, 250 μL Folin–Ciocalteu reagent, 750 μL 20% sodium carbonate and 950 μL DI water. This total of 5 mL solution was incubated for 30 min in 40 °C water base, and then 200 μL solution out of 5 mL was pipetted into a microplate plate with 96 wells (Corning Incorporated, Corning, NY, USA), then determined by absorption at 760 nm in a Synergy H1 microplate reader (BioTek, Winooski, VT, USA). Here, 0.1–1 mg/mL gallic acid (GA) was utilized as a standard for the calibration curve, and the results were expressed as gallic acid equivalents (mg GAE/g DM, mg gallic acid/g of sample). Samples included DOP extracts, DOP dry pastes and freeze-dried powder.
4
Evaluating mutant p53 chaperone stabilization
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To investigate HSP90 chaperone dependent stabilization of different mutp53 proteins, cells were treated with Hsp90 ATPase inhibitors Ganetespib (Synta Pharmaceuticals) or Onalespib (Selleckchem). To determine cell confluency, cells were seeded in 96-wells (Corning) and treated with Onalespib or Ganetespib for 24 h. Confluency was determined using the Celigo Imaging Cytometer and the according software (Nexcelom, Software v5.0.0.0).
5
3D Spheroid Formation Protocol
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1000 cells per well were seeded in ultra-low attachment plates with 96-wells (Corning Incorporated, Kennebunk, ME, USA). Plates were centrifuged at 1000 rpm for 5 min to initiate the formation of 3D spheroid. After 3 days incubation, spheroids were generated and ready for performing experiments.
6
Differentiation of 3D HIE Monolayers
7
HIRRV-Specific IgM Detection Assay
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To detect the specific IgM against HIRRV in serum and gut mucus, 100 µl of prepared virus supernatant was coated into wells of microplates (96-wells, Costar) overnight at 4 °C. After three washes with PBST (PBS containing 0.05% Tween-20), all wells were blocked with 4% BSA at 37 °C for 2 h. Thereafter, serum or mucus samples (1:20 diluted in PBS) from five flounder were incubated for 1.5 h as primary antibodies. FIgM-Mab and alkaline phosphatase-conjugated goat anti-mouse IgG were sequentially added into each well and incubated for 1 h as the secondary and third antibodies, respectively. Following three washes, 100 µl 0.1% (w/v) p-nitrophenyl phosphate (pNPP, Sigma, USA) in 50 mM carbonate-bicarbonate buffer (pH 9.8) containing 0.5 mM MgCl2 was added to each well and incubated at room temperature for 20 min in the dark. The reaction was stopped with 50 µl per well of 2 M NaOH and absorbance was measured with an automatic ELISA reader at 405 nm. Myeloma culture supernatant instead of FIgM-Mab was used as the negative control, and each sample was accessed in three parallels.
8
SARS-CoV-2 RBD Antibody ELISA Assay
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Briefly, polystyrene plates with 96 wells (Costar, USA) were coated with the Mab S7 (10 μg/ml); after blocking and washing RBDf at 5 μg/ml was added to the plate. Serial dilutions of sera were incubated and later, an anti-human IgG-peroxidase conjugate was added. After washing, the substrate solution was added. The reaction was stopped, and an automated ELISA reader recorded the absorbance at 492 nm.
9
ACE2-RBD Binding Inhibition Assay
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Serum pools per group were used in the next humoral immune evaluation experiments. Briefly, polystyrene plates with 96 wells (Costar, USA) were coated with mFc-ACE2 molecule at 5 μg/ml. Later, plates were blocked with skim milk. In unison, serial dilutions of serum pools were incubated with RBD human Fc molecule –HRPO conjugate. Some wells were incubated only with the HRPO conjugate (maximum signal) and the negative control wells, only with the dilution buffer. After washing, the mix was incubated in the plate and a TMB substrate solution was added. An automated ELISA reader recorded the absorbance at 450 nm (A450). The ACE2-RBD binding inhibition percent was defined as (1-A450 (sample) / A450 (maximum signal) *100.
10
Antibody Detection ELISA for Flounder
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ELISA was performed according to the procedure as described above. Specifically, for detecting the total antibody, wells of flat-bottom microplates (96-wells, Costar) were coated with 100 μL/well of previously stored flounder serum from each immune group at 4 °C overnight and then successively incubated with IgM MAb 2D8 as the primary antibody and alkaline phosphatase-conjugated goat-anti-mouse IgG as the secondary antibody. For detecting the specific antibody against S. iniae and recombinant proteins rPDHA1 and rGAPDH, 100 μL live S. iniae (1.0 × 109 cfu/mL) or purified recombinant proteins rPDHA1 and rGAPDH (10 μg/mL) per well were coated and incubated with previously stored flounder serum (1:50) from each immune group as the primary antibody, IgM MAb 2D8 as the secondary antibody, and alkaline phosphatase-conjugated goat-anti-mouse IgG as the third antibody. The serum from the PBS group was used as control. Each serum sample was assessed in triplicate.
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