Whole blood cell cultures were performed to determine the intracellular levels of cytokines. Briefly, whole blood was diluted 1:1 with RPMI-1640 medium, supplemented with penicillin/streptomycin (100 U/100 mg/ml), L-glutamine (2 mM), and HEPES (10 mM) (all from Invitrogen) and distributed in 12-well tissue culture plates (Costar). The cultures were then stimulated with PPD, ESAT-6, CFP-10 or anti-CD3 or media alone in the presence of the costimulatory molecules, CD49d /CD28 at 37° C for 6 hrs. Brefeldin A (10 μg/ml) was added after 2 hours. After 6 hours, centrifugation, washing and red blood cell lysis was performed. The cells were fixed using cytofix/cytoperm buffer (BD Biosciences) and cryopreserved at −80°C. For neutralization experiments, whole blood was cultured in the presence of anti-IL-1R (5 μg/ml), anti-IL-6R (5 μg/ml) and anti TNFR1 (5 μg/ml) (R& D Systems) or isotype control antibody (5 μg/ml) (R&D Systems) at 37°C for 1 h following which PPD was added and cultured for an additional 23 h.
12 well tissue culture plate
Manufactured by Corning
Sourced in United States
12-well tissue culture plates are a type of laboratory equipment used for the in vitro cultivation of cells. They provide a standardized and controlled environment for cell growth and experimentation. Each plate contains 12 individual wells, allowing for multiple experiments or cell lines to be studied simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
L glutamine, by Thermo Fisher Scientific (10 mentions)
Hepes, by Thermo Fisher Scientific (8 mentions)
Cytofix cytoperm buffer, by BD (7 mentions)
Glutamax, by Thermo Fisher Scientific (5 mentions)
Alpha mem, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Tnf α, by R&D Systems (3 mentions)
Gm csf, by Aviva Systems Biology (3 mentions)
Cd207 clone dcgm4, by Beckman Coulter (3 mentions)
Cd34 clone 581, by BD (3 mentions)
Isotype control antibody, by R&D Systems (2 mentions)
Hepes solution, by Thermo Fisher Scientific (2 mentions)
Avicel, by FMC Biopolymer (2 mentions)
Nacho3, by Thermo Fisher Scientific (2 mentions)
Cd28 cd49d, by BD (2 mentions)
Facs lysing solution, by BD (2 mentions)
Fast immune brefeldin a solution, by BD (2 mentions)
Pen strep amp b solution, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Lonza (2 mentions)
61 protocols using 12 well tissue culture plate
1
Intracellular Cytokine Profiling in Whole Blood
2
Infectious Virus Titer Determination by Plaque Assay
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Infectious virus titers were determined by plaque assays as described elsewhere (47 (link), 48 (link), 91 (link)). Briefly, supernatants were serially diluted 10-fold in MEM with 1 μg/ml TPCK-trypsin and were inoculated onto 90% confluent MDCK cell monolayers in 12-well tissue culture plates (Corning Costar, Cambridge, MA). The virus was adsorbed for 1 h at 37°C under 5% CO2 before the addition of 3 ml of overlay. The overlay medium contained 1 part liquid medium containing 10× MEM supplemented with 200 mm l -glutamine (Gibco), HEPES solution (Gibco), 7.5% NaCHO3 (Gibco), penicillin-streptomycin-amphotericin B solution (Gibco), and 1 part 2.4% Avicel (FMC BioPolymer, Philadelphia, PA) in water or 1 part 1% agarose in water. Samples from A/WSN/33 or A/CA/04/2009 wells were incubated at 37°C under 5% CO2 for 3 days. B/Yamagata/16/1988 was incubated at 37°C under 5% CO2 for 5 days to allow for better plaque formation. The overlays were removed, the plates were washed twice with PBS, and the cell monolayers were fixed with acetone-methanol (80:20) for 20 min at RT. Following fixation, the plates were stained with crystal violet as described previously, and viral titers were determined (92 (link), 93 (link)).
3
Intracellular Cytokine Profiling of Whole Blood
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Whole blood cell cultures were performed to determine the frequencies of intracellular cytokine-producing cells. Briefly, whole blood was diluted 1:1 with RPMI-1640 medium, supplemented with penicillin/streptomycin (100 U/100 mg/ml), L-glutamine (2 mM), and HEPES (10 mM) (all from Invitrogen, San Diego, CA) and placed in 12-well tissue culture plates (Costar, Corning Inc., NY, USA). The cultures were then stimulated with SsAg, NIE, PMA/ionomycin (P/I) or media alone in the presence of the co-stimulatory reagent, CD49d /CD28 (BD Biosciences) at 37°C for 6 or 18 h, for intracellular cytokine staining or ELISA respectively. Fast Immune Brefeldin A Solution (10μg/ml) (BD Biosciences) was added after 2 hours. After 6 hours, whole blood was centrifuged, washed using cold PBS, and then 1x FACS lysing solution (BD Biosciences, San Diego, CA, USA) was added. The cells were fixed using cytofix/cytoperm buffer (BD Biosciences, San Diego, CA, USA), cryopreserved, and stored at -80°C until use. For cytokine neutralization experiments (n = 15), whole blood from INF individuals was cultured in the presence of anti-IL-10 (5μg/ml) or anti-TGFβ (5μg/ml) or isotype control antibody (5μg/ml) (R& D Sytems) for 1 h following which NIE and brefeldin A was added and cultured for a further 23 h.
4
Whole Blood Antigen Response Assay
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Whole blood cell cultures were performed to determine the in vitro responses to antigens. Briefly, whole blood was diluted 1:1 with RPMI1640 medium supplemented with penicillin/streptomycin (100 U/100 mg/ml), L-glutamine (2 mM), and HEPES (10 mM; all from Invitrogen) and distributed in 12-well tissue culture plates (Costar). The cultures were then stimulated with ESAT-6, CFP-10, or anti-CD3 or with medium alone in the presence of CD49d/CD28 at 37°C for 18 h. Brefeldin A (10 µg/mL) was added after 12 h. After 18 h, centrifugation, washing, and red blood cell lysis was performed. The cells were fixed using cytofix/cytoperm buffer (BD Biosciences, San Jose, CA) and stored at −80°C. For neutralization experiments, whole blood was cultured in the presence of anti-IL-27 (5 μg/ml), anti-TGFβ (5 μg/ml) (R&D Systems, Minneapolis, MN), anti-PDL-1 (5 μg/ml) (eBiosciences, San Diego, CA), and anti CLTA-4 (5 μg/ml) (Ancell), or isotype control antibody (5 μg/ml) (R&D Systems) at 37°C for 6 h following which PPD was added and Brefeldin A (10 µg/ml) was added after 1 h. The cells were then cultured for a further 16 h.
5
Intestinal Explant Culture and IL33 Quantification
6
Influenza Virus Plaque Assay in MDCK Cells
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MDCK cells were used in plaque assays to determine viral titers [27 (link), 42 (link), 53 (link), 60 (link)]. Briefly, supernatants were serially diluted 10-fold in MEM with 1 ug/ml TPCK-trypsin and inoculated onto 90% confluent MDCK cell monolayers in 12-well tissue culture plates (Costar). The virus was adsorbed for 1h at 37°C/5% CO2 before adding 3 ml of an overlay. Overlay media contained 1-part liquid medium containing: 10x MEM supplemented with 200 mm L-glutamine, HEPES solution, 7.5% NaCHO3, Pen/Strep/Amp B solution (all from Gibco, Waltham, MA), and 1% agarose (Sigma) in water. Samples from A/WSN/33 or A/CA/0409 wells were incubated at 37°C/5% CO2 for 3 days. B/Yamagata/16/1988 infected cells were incubated at 37°C/5% CO2 for 5 days to allow for improved plaque formation. Following incubation, the plates were washed 2x with PBS, and the cell monolayers were fixed with methanol: acetone (80:20) for 20 min at room temperature. Following fixation, the plates were stained with 0.2% crystal violet (Fisher Scientific, Waltham, MA) as described to determine the virus titers [27 (link), 42 (link), 53 (link), 60 (link)].
7
In Vitro Immune Response Assessment
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Whole blood cell cultures were performed to determine the in vitro responses to antigens in a subset of INF (n = 22) and UN (n = 22) individuals and in INF individuals 6 months following treatment. Briefly, whole blood was diluted 1:1 with RPMI1640 medium supplemented with penicillin/streptomycin (100 U/100 mg/ml), L-glutamine (2 mM), and HEPES (10 mM; all from Invitrogen) and distributed in 12-well tissue culture plates (Costar). The cultures were then stimulated with Ss Ag, PPD, or LPS or with medium alone. The hookworm infected samples were stimulated with ES Ag or P/I or with with medium alone at 37°C for 18 h. After 18 h, the supernatants were collected and stored at −80°C until further use.
8
Intestinal Explant Culture for IL-33
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Small pieces of small intestine or colon (~5 mm of mid-part) were isolated, rinsed in HBSS/BSA, weighed, and cultured overnight in 12-well tissue culture plates (Costar) in 1000 μl complete DMEM at 37 °C in an atmosphere containing 5% CO2. After centrifugation to pellet debris, culture supernatants were transferred to fresh tubes and stored at −80 °C. IL-33 was quantified in the supernatant of intestinal explant cultures from V33 and WT mice by enzyme-linked immunosorbent assay (ELISA) according to standard manufacturer’s recommendations (eBioscience) and the results were normalized to the weight of the intestinal explant.
9
Evaluating Antifungal Activity of CXCL10 Expressing Cells
10
Antifouling Potential of Compounds
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Antifouling activity was assessed via inhibition of settlement and metamorphosis of larvae of the Pacific transparent sea squirt (Ciona savignyi) and the blue mussel (Mytilus galloprovincialis). Methods followed those published by Grant et al. [81 (link)]. Adults of both species were collected from coastal populations in the Nelson region of New Zealand, held in a recirculating seawater system (18 ± 1 °C, 33 ± 1 PSU) and fed bulk-cultured Isochorysis galbana until ready to spawn. Larval spawning and rearing procedures followed previously described methods for C. savignyi [82 (link)] and M. gallorprovincialis [83 ]. Competent larvae were diluted in artificial seawater to yield 3 ± 1 larvae/mL. Aliquots of these larval suspensions were added to 12-well tissue culture plates (Corning Co-Star) containing serial dilutions of 1 ranging from 0.1–100 µg/mL. Controls were included, and three replicates were performed in all cases. After 5 days of incubation at 18 ± 1 °C, the number of successfully settled and metamorphosed individuals were counted in each well. Sigmoidal dose-response relationships were explored using R Statistical Software [84 ] to determine whether inhibition had occurred relative to the controls.
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