All experimental procedures conformed to institutional guidelines and were approved by an official committee (Landesamt für Gesundheit und Soziales, Berlin, Germany). Microdissection, preparation and maintenance of mouse neural stem cells (NSC) on 2- to 4-week-old C57Bl/6 mice were conducted as described previously.20 (link) The ability of cells to generate neurons and glia were routinely checked. For cytotoxicity and caspase assays, cells were plated on 96-well flat bottom plates (BD Biosciences, Billerica, MA, USA) and PTX toxicity was induced 24 h after passage of cells. Preparation and culture of hippocampal neurons (HCN) was conducted on C57Bl/6 embryos E14 as published previously.21 (link) Human neuronal progenitor cells were derived from human pluripotent stem cells by dual SMAD inhibition as described before.22 (link) Cell differentiation was confirmed by staining for Nestin, SOX 1 and 2 as well as PAX-6 (A24354, ThermoFisher, Darmstadt, Germany) prior to toxicity analysis. For cytotoxicity and caspase assays, cells were plated on 96-well flat bottom plates (BD Biosciences) and cytotoxicity experiments were conducted with the same protocol as for NSC. MCF-7 and HeLa cell lines were obtained from Sigma-Aldrich (Taufkirchen, Germany; Authentication by STR profiling). Cell culture was performed as described before.23 (link)
96 well flat bottom plate
Manufactured by BD
Sourced in United States, Germany
The 96-well flat-bottom plates are a standard laboratory equipment used for various applications. These plates have a flat-bottom design and contain 96 individual wells, each with a consistent volume capacity. They are commonly used for various assays, cell culture, and sample preparation procedures in research and clinical laboratory settings.
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Lab products found in correlation
Elx800, by Agilent Technologies (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (3 mentions)
Prl tk renilla luciferase vector, by Promega (2 mentions)
Dual glo luciferase assay system, by Promega (2 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (2 mentions)
3h thymidine, by Cytiva (2 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Cell titer blue cell viability assay reagent, by Promega (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
384 well plate, by Corning (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Mcf 7, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Polarstar omega spectrophotometer, by BMG Labtech (2 mentions)
Anti cd3ε, by BD (2 mentions)
Elisa kit, by R&D Systems (2 mentions)
Soluble anti cd28, by BD (2 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
85 protocols using 96 well flat bottom plate
1
Neurodevelopment Stem Cell Protocols
2
Quantifying Cytokine Production in LPL
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To measure spontaneous cytokine production by LPL, LPL were cultured in 96-well flat-bottom plate (Falcon; BD Biosciences) without any stimulation for 24 h at 37 °C under 5% CO2. To measure cytokine production by LPL-T cell, isolated LPL was incubated for 48 h in vitro in 96-well flat-bottom plate coating with anti-CD3ε (10 μg/ml) and soluble anti-CD28 (1 μg/ml) antibodies (BD Biosciences, San Diego, CA). The culture supernatants were then harvested and assayed for cytokine concentration with ELISA kits (R&D Systems) according to the manufacturer’s instruction.
3
Ex Vivo T Cell Cytokine Assay
4
Transfection Optimization for Luciferase Assays
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A double-stranded oligonucleotide containing the SNP of interest (31nt +
restriction enzyme sites) was ordered and annealed as described above. SeeSupplementary Note for
vector preparation details. 3 × 105 Jurkat, HH,
and Daudi cells were nucleofected with 0.9 μg of pGL3-Promoter
vector along with 0.1 μg of pRL-TK Renilla luciferase vector
(Promega) in 16 well strips in a 4D nucleofector with the following protocols
and buffers in 20 μL of total volume: Jurkat, SE buffer, CL-120 protocol;
HH, SE buffer, CL-120 protocol; Daudi SF buffer, CA-137 protocol. After
nucleofection, 180 μL of complete RPMI was added and cells cultured in 96
well flat bottom plates (Falcon). After 48 hours, cells were spun down,
resuspended in 75 of fresh complete media, and luciferase/renilla activity
measured using the Dual-Glo Luciferase Assay System (Promega). Firefly
luciferase activity was expressed as relative luciferase units (RLU) after
correction for Renilla luciferase activity. Data were normalized to those cells
transfected with empty pGL3-Promoter vector. Each dot represents an independent
nucleofection reaction.
restriction enzyme sites) was ordered and annealed as described above. See
vector preparation details. 3 × 105 Jurkat, HH,
and Daudi cells were nucleofected with 0.9 μg of pGL3-Promoter
vector along with 0.1 μg of pRL-TK Renilla luciferase vector
(Promega) in 16 well strips in a 4D nucleofector with the following protocols
and buffers in 20 μL of total volume: Jurkat, SE buffer, CL-120 protocol;
HH, SE buffer, CL-120 protocol; Daudi SF buffer, CA-137 protocol. After
nucleofection, 180 μL of complete RPMI was added and cells cultured in 96
well flat bottom plates (Falcon). After 48 hours, cells were spun down,
resuspended in 75 of fresh complete media, and luciferase/renilla activity
measured using the Dual-Glo Luciferase Assay System (Promega). Firefly
luciferase activity was expressed as relative luciferase units (RLU) after
correction for Renilla luciferase activity. Data were normalized to those cells
transfected with empty pGL3-Promoter vector. Each dot represents an independent
nucleofection reaction.
5
SMYD2 Inhibitor Impacts Cancer Cell Growth
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Cancer cells were seeded into 96-well flat-bottom plates (BD Falcon) at 4000 cells per well, and treated with a SMYD2-specific inhibitor, LLY-507 (≥97%, HPLC, #SML1279, Sigma-Aldrich), at concentration of 0, 3, 5, or 7 μM, and cultured at 37°C under 5% CO2 for 24 h. We also examined growth suppressive effects of LLY-507 at 3 μM concentration in four cancer cell lines, compared without the compound. The growth suppressive effects by knockdown of SMYD2 were assessed after 5 days (rapidly-growing cancer cell lines, SNU449 and SNU475) or 7 days (slowly-growing cancer cell lines, HCT116 and SW480) of transfection with siRNAs. The Cell counting kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used for methyl thiazolyl tetrazolium reaction. After reaction for 2 h, the numbers of cells were counted in a microplate reader at 450 nm.
6
Laser-induced Apoptosis in Regulatory T Cells
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CD4+ T cells were enriched from Foxp3-GFP mouse spleens using a MagniSort Mouse CD4 T-cell Enrichment Kit (eBioscience). Foxp3+ cells were then sorted for using a FACSAria II (BD Biosciences). Sorted cells were stained with 5 μg of anti-CD25-Ce6 or isotype-Ce6 on ice for 25 min. Stained cells were washed and 5 × 104 cells were plated in each well of 96-well flat-bottom plates (FALCON) in RPMI-1640 media (Welgene) with 10% fetal bovine serum (Welgene). Plates were centrifuged for 5 min at 1500 rpm. Cells in each well were irradiated with a 660-nm laser (100 mW/cm2) (Micro Laser Systems) for 5 min. Cell apoptosis after 30 min or 15 h of incubation was measured by detecting surface annexin V (BioLegend) expression by a FACSCalibur (BD Biosciences).
7
MDSC-Mediated Cross-Priming of CD8+ T Cells
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Mice bearing EG.7 tumors inoculated with VSSP alone, and either OVA/VSSP or OVA/polyI:C vaccines, were euthanized and splenocytes stained with mAbs specific for CD11b, Gr1 and SIINFEKL peptide bound to H-2Kb. Splenocytes from EL4 TB mice served as negative controls for the expression of OVA antigen.
For assessing the capacity of splenic MDSCs to achieve an effective cross-priming, antigen-specific CD8+ T cells were isolated from OTI transgenic mice by negative selection using CD8a+ T Cell Isolation Kit II (Miltenyi Biotec) and used as effector cells. Splenic MDSCs isolated from EG.7 and EL4 TB mice, inoculated or not with VSSP, were cultured at 1×106 cells per well with 10 μg/mL of both OVA and VSSP for 24 h, or left untreated. Afterward, MDSCs were washed and cocultured at 1:1 ratio with 1×105 OTI CD8+ cells for 96 h in 96-well flat bottom plates (BD Falcon). BM-DCs previously incubated with OVA, OVA and VSSP or pulsed with SIINFEKL peptide were used as controls. As a measure of CD8+ T cell activation, the expression of CD69 molecule was detected by FACS.
For assessing the capacity of splenic MDSCs to achieve an effective cross-priming, antigen-specific CD8+ T cells were isolated from OTI transgenic mice by negative selection using CD8a+ T Cell Isolation Kit II (Miltenyi Biotec) and used as effector cells. Splenic MDSCs isolated from EG.7 and EL4 TB mice, inoculated or not with VSSP, were cultured at 1×106 cells per well with 10 μg/mL of both OVA and VSSP for 24 h, or left untreated. Afterward, MDSCs were washed and cocultured at 1:1 ratio with 1×105 OTI CD8+ cells for 96 h in 96-well flat bottom plates (BD Falcon). BM-DCs previously incubated with OVA, OVA and VSSP or pulsed with SIINFEKL peptide were used as controls. As a measure of CD8+ T cell activation, the expression of CD69 molecule was detected by FACS.
8
Cytotoxicity Evaluation of Galangin Nanoformulation
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The tested cell lines (200 μL, 1 × 105 cells mL−e) were suspended and cultured in 96-well flat-bottom plates (Falcon, Lincoln Park, NJ, USA). Following 48 h maintenance in the exponential growth phase, the cells were treated for 24 h with free galangin and GAL/β-CD. MTT in PBS (100 µL) was then used to label the well-maintained cells for 10–15 min at 37 °C. Tap water was used to wash excess dye, whereas treatment with 50 µL DMSO for 10 min was applied to dissolve air bubbles. The absorbance of cell cultures was measured by using a microplate reader (ELx 800, Bio-Tek Instruments Inc., Winooski, VT, USA) at 492 nm [29 (link)]. The calculation of the rate of inhibition of cell growth was performed by applying the following equation:
where Abc and Abs refer to the OD values for the control and tested samples, respectively.
where Abc and Abs refer to the OD values for the control and tested samples, respectively.
9
Co-culture of Bone Marrow-Derived Dendritic Cells and T Cells
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At day 9, titrated numbers of immature and matured BMDCs, either derived from BALB/c or transgenic BAC and -WT mice, were co-cultured for 72 h in 96-well flat bottom plates (BD Falcon) with 2 × 106 T cells derived from transgenic BAC, -WT, or BALB/c lymph node cells, respectively. Cell cultures were then pulsed with 1 μCi/well (PerkinElmer) for 8–16 h before they were harvested onto glassfiber filtermates using an ICH-110 harvester (Inotech). Filters were counted in a 1450 microplate counter (Wallac). Additionally, aliquots of cell culture supernatants were cryopreserved for cytokine assays before pulsing with[3H]-thymidine when indicated.
10
Cytokine Profiling of Engineered T Cells
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We employed a previously published protocol.11 (link) Briefly, engineered T cells were stimulated with antigen-expressing tumor cells, for 4 hours at 37°C in 96-well flat-bottom plates (Falcon) at a ratio of 2:1 effector-to-target; SKOV-3 was used to stimulate HER2-CAR T cells, KMS-11 was used to stimulate BCMA-CAR T cells and NALM-6 was used to stimulate CD19-TAC T cells. BD GolgiPlug (BD Biosciences), a protein transport inhibitor, was added to T cells prior to incubation with tumor cells following manufacturer’s guidelines. Following the stimulation period, T cells were stained for surface markers (CD4, CD8) followed by fixation and permeabilization using BD Cytofix/Cytoperm (BD Biosciences) to permit detection of intracellular cytokines. Cytokine secretion was detected by staining of IL-2, TNFα, and IFNγ (BD Biosciences), followed by flow cytometric analysis as described above.
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