HCT116 and U2OS cells were obtained from ATCC (Manassas, VA). MCF10A cells were a kind gift of Daniel Haber (Harvard University, MA). Cells were grown in 384-well plates (CellCarrier Ultra 384; PerkinElmer, Waltham, MA), fixed in 4% paraformaldheyde (PFA) and stained with 4′, 6′-diamidino-2-phenylindole (DAPI). Primary human eosinophils were obtained from healthy donors enrolled under NIH protocol NCT000090662 and purified as previously described (17 (link)). The protocol was approved by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases (NIAID) at the National Institutes of Health (NIH). Eosinophils were plated in Poly-d -Lysine coated 384-well plates (CellCarrier Ultra 384; PerkinElmer, Waltham, MA), then fixed in 4% PFA and stained with DAPI. Cells were plated and stained with DAPI either in biological replicate wells (i.e., cells were plated on different plates on different days) for MCF10A and HCT116 cells, or technical replicate wells (i.e., cells were plated on the same plate, on the same day, but in different wells) for U2OS and primary eosinophils.
Cellcarrier 384 ultra
Manufactured by PerkinElmer
Sourced in United States, United Kingdom
The CellCarrier-384 Ultra is a microplate designed for high-throughput cellular assays. It features a 384-well format with an ultra-thin, clear bottom for optimal imaging performance. The product is intended for use in cell-based experiments and assays requiring a high-density microplate format.
Automatically generated - may contain errors
Lab products found in correlation
Tryptose phosphate broth, by Merck Group (3 mentions)
Non essential amino acids (neaa), by Merck Group (3 mentions)
Operetta, by PerkinElmer (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Foetal bovine serum fbs, by Thermo Fisher Scientific (2 mentions)
Syto 11, by PerkinElmer (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Harmony software, by PerkinElmer (1 mentions)
Opera phenix system, by PerkinElmer (1 mentions)
Opera high content screening microscope, by PerkinElmer (1 mentions)
Deltavision elite microscope, by GE Healthcare (1 mentions)
Safire, by Tecan (1 mentions)
Polybrene, by Merck Group (1 mentions)
11 protocols using cellcarrier 384 ultra
1
Cell Culture Protocols for Microscopy
2
Cell Culture and Immunostaining Protocol
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HCT116 and U2OS cells were obtained from ATCC (Manassas, VA, USA). MCF10A cells were a kind gift of Daniel Haber (Harvard University, MA, USA). Cells were grown in 384-well plates (CellCarrier Ultra 384, PerkinElmer, Waltham, MA), fixed in 4% PFA and stained with DAPI. Primary human eosinophils were obtained from healthy donors enrolled under NIH protocol NCT000090662 and purified as previously described (17) . The protocol was approved by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases (NIAID) at the National Institutes of Health (NIH). Eosinophils were plated in Poly-D-Lysine coated 384-well plates (CellCarrier Ultra 384, PerkinElmer, Waltham, MA), then fixed in 4% PFA and stained with DAPI. Cells were plated and stained with DAPI either in biological replicate wells (i.e., cells were plated on different plates on different days) for MCF10A and HCT116 cells, or technical replicate wells (i.e., cells were plated on the same plate, on the same day, but in different wells) for U2OS and primary eosinophils.
3
Neuronal Co-Culture Protocol in 384-well Plates
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The 384 well plate (CellCarrier-384 Ultra, PerkinElmer, USA) surface for 2D neuronal co-culture, were prepared by incubating the plate with 30 µl of 0.01% Poly-L-Ornithine hydrochloride (PLO, Sigma, P2533) in PBS, for 1 h at room temperature, followed by the 3 times rinsing with DPBS. Then the plates were coated with 30 µl of 3.3 µg/ml Laminin (Invitrogen, 23017-015) solution in PBS, at 37 °C for 1 h or at 4 °C for overnight. For 3D neuronal co-cultures, the same 384 well plates were treated with oxygen plasma (5cc/min, 10 min) just before the cell seeding. This treatment increased the surface hydrophilicity of the plate and confirmed the adherence of the fibrin gel cocktail with the well surface89 .
4
Thawing and Culturing EndoC-βH5 Cells
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EndoC-βH5 cells (Human Cell Design) were thawed and cultured following the manufacturer’s protocol. Cells were seeded 20,000 cells/well into 384-well plates (Perkin Elmer CellCarrier-384 Ultra, 6057300, Seer Green, UK). Reverse transfection was performed as described in Section 2.6 . Media was refreshed 4 days post-seeding, GSIS was performed on day 7 as described in Section 2.4 .
5
In Vitro Anti-Wolbachia Potency Assay
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The anti-Wolbachia potency of a range of BZs was determined in vitro, utilizing an insect cell screening assay as described previously (61 (link)). Briefly, the mosquito (Aedes albopictus) derived cell line (C6/36), stably infected with Wolbachia pipientis (wAlbB) (C6/36 wAlbB) were incubated with a panel of BZ compounds, DOX (all 5 μM, dissolved in DMSO), or 1% DMSO control for 7 d with 2,000 cells per well on a 384-well plate (CellCarrier-384 Ultra; PerkinElmer) in Leibovitz media (Life Technologies) supplemented with 20% FBS (Fisher Scientific), 2% tryptose phosphate broth (Sigma-Aldrich), and 1% nonessential amino acids (Sigma-Aldrich). The end-point readout utilized DNA staining of both the host cell nuclei and intracellular Wolbachia (SYTO11) combined with a high-content imaging system (Operetta; PerkinElmer) and analyzed using the associated Harmony software through a cytoplasm texture analysis.
6
Evaluating Anti-Wolbachia Potency In Vitro
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The anti-Wolbachia potency of doxycycline and rifampicin for use in the PK-PD model was determined in vitro, utilising the routine A·WOL screening assay as described previously57 (link). In brief the mosquito (Aedes albopictus) derived cell line C6/36 (ATCC number CRL-1660), stably infected with Wolbachia pipientis (wAlbB) (C6/36 wAlbB) was incubated with the relevant drugs in a concentration range in order to determine a dose response. The drugs were incubated for 7 days with 2,000 cells per well on a 384 well plate (CellCarrier-384 Ultra, PerkinElmer) in Leibovitz media (Life Technologies™) supplemented with 20% foetal bovine serum (FBS, Fisher Scientific), 2% tryptose phosphate broth (Sigma-Aldrich) and 1% non-essential amino acids (Sigma-Aldrich). The end-point read out utilised DNA staining of both the host cell nuclei and intracellular Wolbachia (SYTO® 11) combined with a high content imaging system (Operetta®, PerkinElmer) and analysed using the associated Harmony® software through a cytoplasm texture analysis.
7
Evaluating Anti-Wolbachia Potency of Doxycycline and Minocycline
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The anti-Wolbachia potency of doxycycline and minocycline for use in the PKPD model was determined in vitro, utilising the routine A·WOL screening assay as described previously50 (link). In brief the mosquito (Aedes albopictus) derived cell line (C6/36), stably infected with Wolbachia pipientis (wAlbB) (C6/36 (wAlbB)) was incubated with the relevant drugs in a concentration range in order to determine a dose response. The drugs were incubated for 7 days with 2,000 cells per well on a 384 well plate (CellCarrier-384 Ultra, PerkinElmer) in Leibovitz media (Life Technologies™) supplemented with 20% foetal bovine serum (FBS, Fisher Scientific), 2% tryptose phosphate broth (Sigma-Aldrich) and 1% non-essential amino acids (Sigma-Aldrich). The end-point read out utilised DNA staining of both the host cell nuclei and intracellular Wolbachia (SYTO®11) combined with a high content imaging system (Operetta®, PerkinElmer) and analysed using the associated Harmony® software through a cytoplasm texture analysis.
8
Irofulven Cytotoxicity Screening in hTERT Fibroblasts
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BJ1/hTERT (hTERT-immortalized normal human foreskin fibroblast) cells were seeded in 384-well plates (CellCarrier-384 Ultra; PerkinElmer) and transfected with siRNAs (silencer select, pool of three siRNAs per one gene, final siRNA concentration: 3 nM per well; Thermo Fisher Scientific) using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific). 24 h after transfection, cells were treated with either: (1) 2 μg/ml irofulven for 1 h and cultured for another 4 d without irofulven or (2) with 75 ng/ml irofulven for 4 d. Cells were fixed with 4% paraformaldehyde and stained with 1 μg/ml Hoechst 33342 (Thermo Fisher Scientific) in PBS. The proportions of stained cells were determined using an Opera Phenix system and Harmony software (PerkinElmer).
9
Quantifying Heterogeneous Cell Populations
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Single colonies of recombinants obtained as described above were inoculated into 50 μL MSA medium supplemented with 100 mg/l adenine and grown for two days at 30°C in 96 well plates. Cells were then diluted 24 times into MSA medium with adenine and grown for ~20 hr at 30°C. Sixty μl cell suspensions diluted 15 times with MSA medium with adenine were transferred to 384 well microplates with clear bottom (CellCarrier-384 ultra, Perkin Elmer). The fluorescence of cells was measured using an Opera high-content screening microscope (Perkin Elmer). The following settings were used [Filter sets: Camera 1: 475/50 for CFP signals, Camera 2: 540/75 for YFP signals, Camera 3: 690/50 for bright field, Light source: 405/488/635]. Twelve images were taken for each well, for a total of ~200 cells per strain. The proportion of P cells was then calculated using the Acapella software program (PerkinElmer). Selected strains were imaged again using a Delta Vision Elite microscope (GE Healthcare).
10
Ratiometric Autophagy Flux Assay in PC12D Cells
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For ratiometric autophagy flux assay, PC12D-GFP-LC3-RFP cells were seeded into a 384-well black plate (CellCarrier 384-Ultra; PerkinElmer, 6057308). After 72 h differentiation by exposure to 100 ng/mL NGF, cells were treated with compounds for 24 h. Cells were then fixed with 10% formalin containing 2 µg/mL Hoechst33342 (Invitrogen, H3570) for 30 min. Image capture and quantification of GFP and RFP intensity in cells were performed using a high-content imager, OPERA Phenix and Harmony software ver 4.5 (PerkinElmer, Waltham, MA), or a plate-reader, SAFIRE (TECAN, Männedorf, Switzerland). For the autophagy flux assay using fluorescence imaging with tfLC3, NGF-differentiated PC12D cells were transfected with the tfLC3 vector. At 48 h after transfection, cells were treated with the indicated compounds for 8 h. Fixation, confocal microscopy was then performed as previously described [80 (link)].
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