Generated stable anti-polyST IBs and luciferase expressing TE671 cell lines were incubated with anti-polySia 735 [85 (link)] or mouse anti-NCAM ERIC1 (Santa Cruz Biotechnology) antibodies to determine polySia and NCAM surface expression by flow cytometry. Staining with antibodies was performed for 30 min at 4 °C in a 96-well microtitre plate (Nunclon™ Surface plate, Nunc) in 100 μl PBS containing 2% FCS (Invitrogen). For detection of the primary antibodies an goat anti-mouse RPE-labeled antibody (Dianova) was used. As negative controls an incubation step with endosialidase [84 (link)] before applying the primary detection antibody was performed or the primary anti-NCAM antibody was not used. Cells were washed once with PBS containing 2% FCS and resuspended in 300 μl PBS containing 2% FCS and 10 μg/ml propidiumiodide for subsequent analysis using a FACS-calibur equipped with CellQuest software (Becton Dickinson).
Fetal calf serum (fcs)
Manufactured by BD
Sourced in United Kingdom, United States
The FCS is a piece of lab equipment designed for fluorescence correlation spectroscopy. It measures the fluctuations in fluorescence intensity to determine the diffusion and concentration of fluorescently labeled molecules in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Fetal calf serum (fcs), by Merck Group (5 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (5 mentions)
Facsaria 3, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Dimethyl sulfoxide (dmso), by BD (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Facs lysing solution, by BD (1 mentions)
Rpmi 1640 media, by Lonza (1 mentions)
Las x software, by Leica camera (1 mentions)
Crl 2768, by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Penicillin, by BD (1 mentions)
Streptomycin, by BD (1 mentions)
Glass insert, by MatTek (1 mentions)
Sepmate 50 tube, by STEMCELL (1 mentions)
Ficoll paque premium, by GE Healthcare (1 mentions)
Cellquest software, by BD (1 mentions)
Dulbecco modified eagle medium (dmem), by BD (1 mentions)
14 protocols using fetal calf serum (fcs)
1
Quantification of PolySia and NCAM Expression
2
Isolation and Characterization of Dsg3-specific B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood using Lymphocyte Separation Medium (Capricorn, Ebsdorfergrund, Germany). Mouse BCH clones were cultured in RPMI-1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine (all Capricorn) and 10% FCS (Merck Millipore, Berlin, Germany). PBMC were washed twice with PBS + 1% FCS and 1 ×106 cells per sample were subsequently stained with Dsg3-AF647 together with mouse anti-human CD19-PerCP-Cy5.5 (HIB19), mouse anti-human CD27-PE (M-T271), mouse anti-human CD38-FITC (HIT2) and the respective isotype controls (all BD Biosciences, Heidelberg, Germany) or with goat anti-mouse IgG-AF488 (A-11029; Thermo Fisher Scientific, Waltham, MA, USA) for mouse BCH clones. After incubation for 20 min at 4°C cells were washed twice with PBS + 1% FCS and a minimum of 2.5 ×105 PBMC or 0.5 ×105 BCH per sample were acquired on a FACS Calibur (BD Biosciences). In a subset of experiments, sorting of cells was performed using FACS Aria III (BD Biosciences). Data analysis was performed using FlowJo 7.6 (TreeStar Inc., Ashland, USA).
3
Whole Blood Immunological Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells from adults were isolated by density gradient centrifugation (Ficoll histopaque, Lonza) from blood collected in sodium (Na)-heparin tubes (Greiner Bio-one) or heparinized blood bags. PBMC were analyzed fresh or cryopreserved in RPMI 1640 media (RPMI, Lonza) with 10% v/v dimethyl sulfoxide (DMSO, Sigma-Aldrich) and 45% v/v fetal bovine serum (Biochrom).
Whole blood intracellular cytokine staining (WB-ICS) assays were performed as described previously (26 (link)–28 (link)). Briefly, 1 mL whole blood was either left unstimulated (negative control) or stimulated with phytohemagglutinin (at 10 μg/mL, positive control), peptide pools of Ag85B, ESAT-6, or CFP-10 (all 15mer peptides, overlapping by 10 aa at 2 μg/mL, GenScript) or BCG (≈1.2 × 106 CFU/mL, Statens Serum Institut) for 12 h or 7 days (BCG only, used at 1 × 105 CFU/mL). Thereafter, red cells were lysed and white cells fixed using FACS-Lysing solution (BD Biosciences), before cryopreservation in 10% DMSO in fetal calf serum.
Whole blood intracellular cytokine staining (WB-ICS) assays were performed as described previously (26 (link)–28 (link)). Briefly, 1 mL whole blood was either left unstimulated (negative control) or stimulated with phytohemagglutinin (at 10 μg/mL, positive control), peptide pools of Ag85B, ESAT-6, or CFP-10 (all 15mer peptides, overlapping by 10 aa at 2 μg/mL, GenScript) or BCG (≈1.2 × 106 CFU/mL, Statens Serum Institut) for 12 h or 7 days (BCG only, used at 1 × 105 CFU/mL). Thereafter, red cells were lysed and white cells fixed using FACS-Lysing solution (BD Biosciences), before cryopreservation in 10% DMSO in fetal calf serum.
4
P0 Specificity of Cy5-P0 Peptide Derivatives
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The P0 specificity of Cy5-P0101–125 was previously determined in P0-expressing myelinating Schwannoma cells (RT4 D6P2T myelinating Schwannoma cells (ATCC® CRL-2768™; [34 (link)])) and non-P0-expressing cells, using P0 antibodies and a fluorescent derivative of the extracellular portion of P0 [27 (link)]. P0-expressing RT4 D6P2T cells were grown in Dulbecco’s modified Eagle medium (Life Technologies, Paisley, UK) containing penicillin, streptomycin, and fetal calf serum (All BD Biosciences) at 37 °C and 5% CO2. Cells were trypsinized and seeded onto 35 mm culture dishes that contained a glass insert (MatTek co) one or two days prior to the imaging experiment. One hour prior to imaging, 1 μM Cy5-P101–125, Cy5-P0101–120, Cy5-P0101–115, Cy5-P0101–110, Cy5-P0101–106, Cy5-P0105–125, Cy5-P0108–125, Cy5-P0112–125, Cy5-P0116–125, Cy5-P0120–125, or the original lead compound Cy5-P0101–125 [27 (link)] was added (incubation at 4 °C; N = 3 per tracer). Imaging was performed as previously described [35 (link)]. Image acquisition and processing were performed using LASX software (Leica Application Software Suite 4.8). Image analysis, including (3D) surface plot and co-localization analysis, was performed using Image J [26 (link)].
5
Isolation and Cryopreservation of PBMC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood samples collected from buffy coats from anonymized blood donors at Uppsala University hospital were used to enrich peripheral blood mononuclear cells (PBMC). PBMC were enriched using Ficoll-Paque Premium (ρ=1.076 g/ml) (GE Healthcare, Little Chalfont, UK) in SepMate™-50 tubes (Stemcell Technologies, Vancouver, Canada). Platelets were removed by centrifugation (2 x 200g, 10 minutes). Enriched PBMC were frozen and kept at -80 °C in heat inactivated fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA) with 10% of DMSO. On the day of the cell sorting the enriched cryopreserved PBMCs were after thawing incubated in PBS, pH 7.4 with 2% heat inactivated fetal calf serum containing the following fluorescent-conjugated antibodies targeting CD4 (RPA-T4), CD8 (RPA-T8), CD14 (M5E2), CD19 (HIB19) from BD Bioscience and eBioscience, San Diego, CA, USA. For the Ampliseq analysis, the cells (106 cells of each population) were collected in PBS, pH 7.4 with 2% heat inactivated fetal calf serum and kept on ice until RNA extraction. The cell sorting was performed on a FACSAria III (BD Biosciences). Data analysis was performed using FlowJo software version 9.8.
6
Isolation and Culture of Human Skin Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human skin fibroblasts were isolated from 3-mm skin punch biopsy (n = 15 for RTT and n = 15 for controls). Cells were cultured in DMEM (Sigma-Aldrich, Milan, Italy), containing 20% fetal calf serum [9] (link) (FCS, Sigma-Aldrich) and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin) (Lonza, Milan, Italy). Cells were incubated at 37 C° in a humidified atmosphere at 5% CO2 for 3 days. When fibroblasts growing from the dermal pieces formed a confluent layer, the dermal pieces were removed and trypsin/EDTA mixture (Sigma-Aldrich) was added to separate fibroblasts. Cells were transferred to 25-cm2 culture flasks (Falcon, Perugia, Italy) and subcultured in 10% FCS/DMEM. Fibroblasts from passage 3 to 5 were used for the experiments. 1×106 cells were seeded in each flask (25 cm2), whereas the experiments were performed when cells reached 70/80% confluence.
7
Isolation of Schwann and Myofibroblast Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Schwann and myofibroblast cells were isolated from tumor specimens according to Eduard Serra Protocol [36 (link)]. In brief, tumor pieces were shredded and then digested in DMEM, 10% FCS (Gibco BRL, Paisley, UK), 500 U/mL penicillin/streptomycin (Sigma-Aldrich), 160 U/mL collagenase type 1 (Sigma-Aldrich), and 1 U/mL dispase grade 1 (Sigma D4818). Following an incubation period of 20 h at 37 °C and 10% CO2 in this buffer, samples were further melted by pipetting with a narrowed Pasteur pipette. The suspension was moved to a 50 mL Falcon tube with DMEM with 10% FCS, centrifuged at 3000× g for 10 min, and suspended in a fresh buffer composed of DMEM, 10% FCS, 500 U/mL penicillin/streptomycin, 0.5 mM 3-iso-butyl-L-methylxanthine (IBMX; Sigma-Aldrich), 10 nM β1-heregulin (Peprotech, #100-03, London, UK), 0.5 µM forskolin and 2.5 µg/mL insulin (Sigma-Aldrich). Cells were seeded at a density of 25 000 cells/cm2 onto six-well plates (Falcon, Milville, NJ, USA), precoated with 1 mg/mL poly-L-lysine (Sigma-Aldrich) and 4 µg/mL natural mouse Laminin (Sigma-Aldrich). Cultures were incubated in a humidified atmosphere at 37 °C and 10% CO2. The medium was changed twice a week and cells were passed when cultures were confluent (usually after 5–7 days).
8
Intestinal Epithelial Cell Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After Peyer’s patches and connective tissues were removed from small intestines, they were opened longitudinally, washed three times in ice-cold PBS buffer to clean their contents, then cut into 0.5cm pieces and incubated with gentle stirring for 30min at 37°C in RPMI 1640 containing 1% FCS (Gibco) and 1mM EDTA. After incubation, tubes were shaken vigorously and the content was passaged through a 100 µm cell strainer (BD Falcon), centrifuged at 400 × g for 5min and washed with PBS buffer containing 2% FCS. Then cell pellets were resuspended in 5ml of 40% Percoll, and centrifuged at 2000 × g for 10min. The resulting cell pellet was washed two times with PBS containing 2% FCS and used for flow cytometry analysis.
9
Primary Mixed Rat Cortical Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary mixed cultures were prepared from the cortices of day 17-18 (E17-18) Wistar rat fetuses, as described previously (Brewer, 1995) with minor modifications. The cortex was dissected out and individual cells were disaggregated mechanically in HBSS (Invitrogen) containing 0.25% trypsin (Invitrogen) and 4 mg/ml DNase (Sigma). The cells were plated in poly-D-Lysine coated plates (Falcon) at a density of 1.5 × 10 6 per 6 cm, and incubated for 4 h in Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS (Fetal Calf Serum), 10 mM glucose, 10 mM sodium pyruvate, 0.5 mM glutamine, 0.05 mg/ml gentamicin, 0.01% streptomycin and 100 μU/ml penicillin G. Finally, this medium was replaced with Neurobasal/B27 culture medium containing 0.5 mM glutamine. Cytosine arabinoside was not included in the culture medium to allow glial proliferation and the cultured cells were used in the second week after plating. The relative abundance of astrocytes and neurons at this stage was determined by immunostaining of their respective markers, GFAP and MAP2. Immunostaining methods have been previously described (Gonzalez-Gonzalez et al., 2008) .
10
Precursor B Cell Leukemia NALM-6 Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human B cell precursor leukemia cell line NALM-6 (ACC128) was purchased from DSMZ (German collection of microorganisms and cells, Braunschweig, Germany) and maintained in RPMI-1640 supplemented with 2 mM l -glutamine (Life Technologies GmbH, Darmstadt, Germany) and 10% FCS (Biochrom AG, Berlin, Germany). HEK293T cells were maintained in DMEM (Life Technologies) supplemented with 2 mM l -glutamine and 10% FCS. PDX cells were cultured in RPMI medium supplemented with 20% FSC, 1% pen/strep, 1% gentamycin, 6 mg/l insulin, 3 mg/l transferrin, 4 μg/l selenium (Gibco, San Diego, USA), 2 mM glutamine, 1 mM sodium pyruvate, 50 μM α-thioglycerol (Sigma-Aldrich, St. Louis, USA). For drug treatments, Vincristine (VCR, Merck, Darmstadt, Germany), Etoposide (Merck) or Methotrexate (Merck) was added to the culture medium. Cell viability was detected by LSRII (BD Bioscience, San Jose, CA) based on FCS/SSC gating at the end of the experiment.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!