Murine colon carcinoma cells (CT26 and MC38) as well as the murine pancreatic carcinoma cells (PDA6606) and human non-malignant keratinocytes (HaCat) were maintained in Roswell Park Memorial Medium (RPMI) 1640 (Pan Biotech), containing 10% fetal bovine serum, 2% glutamine, and 1% penicillin-streptomycin (all Sigma). Cells were incubated at 37 °C, 95% humidity, and 5% CO2. Sub-culturing was performed twice a week. For experiments, 1 × 104 cells were seeded in flat-bottom 96-well plates (Eppendorf). These plates have a rim, which can be filled with sterile water to prevent excessive evaporation during incubation and subsequent alterations in the outer wells of the plate (edge effects).
96 well flat bottom plates
Manufactured by Eppendorf
Sourced in Germany
The 96-well flat-bottom plates are a common laboratory equipment used for various applications. These plates provide a standardized format with 96 individual wells arranged in an 8x12 grid, each with a flat bottom. The plates are designed to accommodate small volumes of liquid samples or reagents for high-throughput experiments and assays.
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Lab products found in correlation
Penicillin streptomycin, by Merck Group (2 mentions)
Pam3csk4, by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Glutamine, by Merck Group (1 mentions)
Rpmi 1640, by PAN Biotech (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Immunocult human cd3 cd28 t cell activator, by STEMCELL (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Purified human c1q, by Complement Technology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Infinite f50, by Tecan (1 mentions)
Phc0045, by Thermo Fisher Scientific (1 mentions)
Cell culture incubator, by Binder (1 mentions)
Penicillin streptomycin, by Eppendorf (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by PAN Biotech (1 mentions)
8 protocols using 96 well flat bottom plates
1
Culturing Murine and Human Cell Lines
2
In vitro Anti-C1q Autoimmunity Assay
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The in vitro model of anti‐C1q autoimmunity was used as described before.32 , 84 , 85 Briefly, flat‐bottom 96‐well plates (Eppendorf, Hamburg, Germany) were coated with 70 μL of 5 μg mL−1 purified human C1q (Complement Technology, Tyler, TX, USA) in coating buffer (0.4 m sodium carbonate buffer, pH 9.6) overnight at 4°C. The plates were washed twice with 140 μL PBS (Life Technologies) before adding anti‐C1q‐positive (SLE) or anti‐C1q‐negative (NHS) sera. Each serum sample was centrifuged at 14 000 g at 4°C for 30 min and diluted at 1:100 in PBS 1 m NaCl (Sigma‐Aldrich, St. Louis, MO, USA) before incubation on a shaker (500 rpm) at room temperature for 1 h. Again, plates were washed four times with 140 μL PBS. Next, PBMCs (200 000 per well), T cells (200 000 per well) or monocytes/T cells (20 000 monocytes and 100 000 T cells per well) were added and activated with 5 μL mL−1 soluble tetrameric anti‐CD3/anti‐CD28 complex (ImmunoCult™ Human CD3/CD28 T cell activator; Stemcell Technologies) in a volume of 200 μL for 24 h.
3
Cryopreserved PBMC Stimulation Assay
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Cryopreserved PBMC samples were rapidly thawed in a 37°C water bath, transferred to 15 ml tubes containing ~ 3 ml RPMI+10% FBS and centrifuged at 800g for 5 min at RT. A total of 200000 cells resuspended in 200 μL culture medium [RPMI-1640 (GIBCO, Invitrogen) supplemented with 10% FBS (GIBCO), 100 U/ml penicillin and 100 μg/ml streptomycin, (SIGMA)] were seeded per well in 96-well flat-bottom plates (Eppendorf) and stimulated with either 106 cfu/ml of heat-killed Candida albicans Strain SC5314 (kind gift from David Moyes; King’s College London), 0.2 × 106 cfu/ml of BCG (TUBERVAC, Serum Institute of India), 50 μg/ml Pam3CSK4 (Sigma) or 1ng/ml LPS (Sigma). Cells cultured with medium alone were used as negative control. Cells were cultured for 24 hr after which plates were centrifuged at 800g for 3 minutes and culture supernatants were collected and frozen at −20°C till ELISA was performed.
4
Cytotoxicity Evaluation of Phytogenic Substances
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The neutral red (NR ) cell viability assay (Aniara Diagnostica, West Chester, Ohio, USA), which targets lysosomal activity, was performed to study the cytotoxic influence of the phytogenic test substances on IPEC-J2 and to define nontoxic test concentrations for further experiments. IPEC-J2 were seeded at a density of 3 × 104 cells/well in 96-well flat-bottom plates (Eppendorf, Hamburg, Germany) and cultured for 24 h to reach approximately 100% confluence. The supernatants were discarded and the cells were treated with 200 µL of either complete growth medium (cell control [CC ]), various concentrations of the phytogenic substances, or 100 µg/mL TYL for 24 h. On the next day, the NR cell viability assay was performed according to the manufacturer’s instructions. Briefly, cells were washed and incubated with a 1:100 NR solution (diluted in complete growth medium) for 3 h, allowing accumulation of the dye in the lysosomes. Subsequently, the cells were fixed for 1 min, the incorporated dye was dissolved, and the absorbance was measured at 540 nm with a reference wavelength of 690 nm.
5
Cytotoxicity Assessment of Memristor Materials
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To assess the cytotoxicity of materials used in the memristor device, a series of samples coated with sputtered Au, as well as samples coated with TiO2 ink were prepared separately and placed into clear 96-well flat-bottom plates (Eppendorf). An additional empty plate was reserved for the control test. The cells of neuroblastoma (IMR-32, ATCC® CCL-127™, USA) and postnatal human fibroblast (PHF, Institute of Cytology RAS, Russia) lines were taken each in a quantity of 5 × 103 and seeded in the prepared 96-well plates with the samples, as well as in the control one. The cell viability was measured using the standard methylthiazol tetrazolium (MTT) assay.42 (link) In brief, the cells were incubated for 72 h at 37 °C. The incubated product was tested by adding 0.2 mL of MTT (5 mg mL−1) for 2 hours, then the MTT-formazan product was dissolved in 0.2 mL of DMSO and the optical absorbance was measured at 570 nm using a plate reader Infinite F50 (Tecan). To improve cells visibility, trypan blue solution (0.4 wt%) was added as cell stain. The experimental reproducibility was provided with three individual batches prepared for each of the experimental tests.
6
Evaluating CD40-CEACAM5 Bispecific Antibodies
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Example 8
Aim and Background
The aim of this study was to assess the effect of the CD40-CEACAM5 bispecific antibodies on B cell activation in vitro in the presence or absence of CEACAM5. CD40 crosslinking will be mediated by simultaneous binding of CD40, expressed on B cells, and CEACAM5 transfected CHO cells.
Materials and Methods
The agonistic effect of CD40-CEACAM5 bispecific antibodies was assessed in a B cell assay, based on primary human B cells. Briefly, B cells were isolated from human peripheral blood mononuclear cells by MACS according to the manufacturer's protocol (Miltenyi Biotec #130-091-151). Human CEACAM5 transfected CHO cells, cynomolgus CEACAM5 transfected CHO cells or CHO wt cells were UV irradiated and seeded in tissue culture treated 96 well flat bottom plates (Eppendorf). B cells were cocultured with the CHO cells in the presence of IL-4 (10 ng/ml, Gibco #PHC0045) and titrated concentrations of CD40-CEACAM5 bispecific. After 2 days, B cells were harvested and expression level of the activation marker CD86 was analyzed by FACS.
Results and Conclusions
The data demonstrate that tested CD40-CEA RUBYs induce upregulation of CD86 on B cells in the presence of CEA (
7
Cryopreserved PBMC Stimulation Assay
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Cryopreserved PBMC samples were rapidly thawed in a 37°C water bath, transferred to 15ml tubes containing ~ 3ml RPMI+10% FBS and centrifuged at 800g for 5min at RT. A total of 200000 cells resuspended in 200µL culture medium [RPMI-1640 (GIBCO, Invitrogen) supplemented with 10% FBS (GIBCO), 100U/ml penicillin and 100µg/ml streptomycin, (SIGMA)] were seeded per well in 96-well flat-bottom plates (Eppendorf) and stimulated with either 106 cfu/ml of heat-killed Candida albicans Strain SC5314 (kind gift from David Moyes; King’s College London), 0.2 x 106 cfu/ml of BCG (TUBERVAC™, Serum Institute of India), 50μg/ml Pam3CSK4 (Sigma) or 1ng/ml LPS (Sigma). Cells cultured with medium alone were used as negative control. 24hr later plates were centrifuged at 800g for 3min and culture supernatants collected and frozen at -20°C till ELISA was performed.
8
Culturing Pancreatic Cancer Cell Lines
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Pancreatic cancer cell lines (MiaPaCa2, PaTuS, PaTuT, and Panc01) were cultured in Dulbecco’s modified Eagle’s Medium (DMEM, Pan Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FCS), 2% glutamine, and 1% penicillin/streptomycin (all Sigma, Steinheim, Germany). For incubation, cells were placed at 37 °C and 5% CO2 in a humidified cell culture incubator (Binder, Tuttlingen, Germany). For in-vitro experiments with 2D cell cultures, 2 × 104 cells were seeded in 100 µL of Roswell Park Memorial Medium (RPMI, Pan Biotech) also supplemented with FCS, glutamine, and penicillin-streptomycin, in tissue culture-treated 96-well flat-bottom plates (Eppendorf, Hamburg, Germany). Cell counting was performed in a highly standardized fashion by determining the absolute number of cells using the Attune NxT flow cytometer (Thermo Scientific, Waltham, MA, USA) and propidium iodide (PI; Sigma) for live-dead discrimination. For optimal culture conditions, the rim of the Eppendorf plates was filled with double-distilled water to prevent excessive evaporation of culture medium in the outer wells (edge effect).
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