Expression of tumor antigens on tumor cells was analyzed by flow cytometry. Tumor cells (1.0 × 106) were harvested and first incubated with 1 μl per test of LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA, USA) in 1× phosphate-buffered saline (PBS) for 30 min at 4°C to accomplish live vs. dead cell discrimination. Cells were then centrifuged, washed twice with cold PBS, and then stained in 1× PBS + 1% BSA (Teknova, Hollister, CA, USA) for 30 min at 4°C with the following anti-human mAbs: Pacific Blue-conjugated or PE-conjugated NEO-201 antibody (BioLegend, San Diego, CA, USA), CEACAM5-FITC (clone C365D3), CEACAM6-PE (clone KOR-SA3544; ThermoFisher Scientific, Waltham, MA, USA). After staining, cells were washed twice with cold PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). Analysis of cellular fluorescence was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). Positivity was determined using fluorescence-minus-one controls.
Bovine serum albumin (bsa)
Manufactured by Teknova
Sourced in United States
BSA (Bovine Serum Albumin) is a commonly used laboratory reagent that serves as a protein source and stabilizer. It is derived from bovine blood serum and is widely used in various biological applications, such as cell culture, immunoassays, and protein purification.
Automatically generated - may contain errors
Lab products found in correlation
Live dead fixable aqua, by Thermo Fisher Scientific (4 mentions)
Facsverse flow cytometer, by BD (4 mentions)
Facsuite software, by BD (4 mentions)
Pacific blue conjugated neo 201 antibody, by BioLegend (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Sodium azide, by Teknova (1 mentions)
Phosphate buffered saline (pbs), by Rockland Immunochemicals (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Hoefer (1 mentions)
Sulfuric acid, by Thermo Fisher Scientific (1 mentions)
Immulon 4 hbx elisa plates, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Spectramax i3, by Molecular Devices (1 mentions)
Human trustain fcx, by BioLegend (1 mentions)
Cd56 pe, by BioLegend (1 mentions)
Tim 3 pe cy7, by BioLegend (1 mentions)
Granzyme b fitc clone gb11, by BioLegend (1 mentions)
Nkg2d bv421, by BioLegend (1 mentions)
Pd 1 apc clone eh12.2h7, by BioLegend (1 mentions)
7 protocols using bovine serum albumin (bsa)
1
Tumor Antigen Expression Analysis
2
PBMC Preparation and Staining for CyTOF
3
Quantification of C1q Binding
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Immulon 4HBX ELISA plates (Thermo Fisher Scientific) were coated overnight with bovine serum albumin (BSA, Thermo Fisher Scientific), ALX217, ALX148, or ALX222 at 5 μg/ml in PBS. Plates were blocked in assay buffer pH 6.0 (PBS with 0.5% BSA, 0.05% Tween-20, 0.25% CHAPS, 5mM EDTA, 0.35M NaCl, Teknova) and washed in 1x TBST (Teknova). Plates were incubated with complement C1q (Quidel) for 1 hour at room temperature and washed with wash buffer. Plates were then incubated for 1 hour at room temperature with HRP-conjugated sheep anti-complement C1q antibody (Thermo Fisher Scientific) at 2 μg/mL and washed, followed by the addition of 3,3’,5,5’-tetra-methylbenzidine peroxidase substrate (Thermo Fisher Scientific) and incubation for 10 minutes. The reaction was terminated with 0.16M sulfuric acid (Thermo Fisher Scientific) and the resulting absorbance was measured at 450 nm with a reference of 570 nm using a SpectraMax i3 plate reader (Molecular Devices).
4
Flow Cytometric Analysis of O-glycan Expression
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Cells to be profiled for the expression of O-glycans were selected for their reactivity with NEO-201 in flow cytometry. CFPAC-1, HL-60, U937 and K562 cell lines and human neutrophils were chosen as NEO-201 target cells.
The cells were first incubated with 1 μL per test of LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA, USA) in 1 mL of 1x phosphate buffered saline (PBS) (VWR International, Radnor, PA, USA) for 30 min at 4 °C to accomplish live-versus-dead-cell discrimination. Then, the cells were washed with 1x PBS and incubated with 2–5 μL of Human TruStain FcX™ (BioLegend, San Diego, CA, USA) in 100 uL of 1× PBS at room temperature for 5–10 min. To evaluate the reactivity of the cells with NEO-201, the cells were then stained in 100 μL of 1× PBS + 1% BSA (Teknova, Hollister, CA, USA) for 30 min at 4 °C with Pacific Blue conjugated NEO-201 (BioLegend, San Diego, CA, USA).
After staining, the cells were washed twice with cold 1× PBS and examined using an FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). The analysis of cellular fluorescence was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). Positivity was determined by comparing unstained cells with cells stained with NEO-201. Staining values > 10% were considered positive.
The cells were first incubated with 1 μL per test of LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA, USA) in 1 mL of 1x phosphate buffered saline (PBS) (VWR International, Radnor, PA, USA) for 30 min at 4 °C to accomplish live-versus-dead-cell discrimination. Then, the cells were washed with 1x PBS and incubated with 2–5 μL of Human TruStain FcX™ (BioLegend, San Diego, CA, USA) in 100 uL of 1× PBS at room temperature for 5–10 min. To evaluate the reactivity of the cells with NEO-201, the cells were then stained in 100 μL of 1× PBS + 1% BSA (Teknova, Hollister, CA, USA) for 30 min at 4 °C with Pacific Blue conjugated NEO-201 (BioLegend, San Diego, CA, USA).
After staining, the cells were washed twice with cold 1× PBS and examined using an FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). The analysis of cellular fluorescence was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). Positivity was determined by comparing unstained cells with cells stained with NEO-201. Staining values > 10% were considered positive.
5
Amplification and Labeling of Biomolecules
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For initial experiments, clones of selected particles were generated via PCR of the pGEM T-easy insert, followed by asymmetric PCR to amplify the template strand. Circularization and RCA were then performed, as above. RCA conditions were 30°C for 30 m with 3 nmol dNTP, followed by heat inactivation for 10 m at 65°C—these are the standard RCA conditions used, unless otherwise noted. Templates for clones of interest were synthesized (IDT).
All stainings were performed in a pre-blocked 96-well v-bottom plate. Pre-block was PBS 1% bovine serum albumin (BSA) (Sigma, St Louis, MO, USA) supplemented with 10 mM MgCl2 (Teknova, Hollister, CA, USA; PBS 1% BSA 10 mM MgCl2). Unless otherwise noted, ∼3 × 1010 fluorescently-labeled particles were incubated with 2 μl coated beads for 20 m at RT in PBS 10 mM MgCl2. Beads were then washed once with PBS 10 mM MgCl2, twice by Tris-buffered saline (Mediatech) 0.05% Tween-20 (Thermo Fisher Scientific) 10 mM MgCl2 (TBST 10 mM MgCl2), once with PBS 10 mM MgCl2 and resuspended in PBS 10 mM MgCl2. Washes were performed by magnetic pulldown (streptavidin beads) or centrifugation at 1000 × g for 3 m (rituximab and bevacizumab beads). Fluorescence was measured with a multimode microplate reader (TECAN, Männedorf, Switzerland).
All stainings were performed in a pre-blocked 96-well v-bottom plate. Pre-block was PBS 1% bovine serum albumin (BSA) (Sigma, St Louis, MO, USA) supplemented with 10 mM MgCl2 (Teknova, Hollister, CA, USA; PBS 1% BSA 10 mM MgCl2). Unless otherwise noted, ∼3 × 1010 fluorescently-labeled particles were incubated with 2 μl coated beads for 20 m at RT in PBS 10 mM MgCl2. Beads were then washed once with PBS 10 mM MgCl2, twice by Tris-buffered saline (Mediatech) 0.05% Tween-20 (Thermo Fisher Scientific) 10 mM MgCl2 (TBST 10 mM MgCl2), once with PBS 10 mM MgCl2 and resuspended in PBS 10 mM MgCl2. Washes were performed by magnetic pulldown (streptavidin beads) or centrifugation at 1000 × g for 3 m (rituximab and bevacizumab beads). Fluorescence was measured with a multimode microplate reader (TECAN, Männedorf, Switzerland).
6
Flow Cytometry Analysis of NK Cells and Carcinoma Lines
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Analysis of the expression of cell surface and intracellular proteins in purified NK cells and in human carcinoma cell lines was performed by flow cytometry. Cells (1.0 × 106 (link)) were incubated with 1 μL per test of LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA) in 1 × phosphate buffered saline (PBS) for 30 min at 4°C to accomplish live versus dead cell discrimination. Cells were then centrifuged, washed twice with cold PBS, and then stained with primary antihuman mAbs in 1 × PBS +1% BSA (Teknova, Hollister, CA) for 30 min at 4°C. Binding of NEO-201 to human carcinoma cell lines was detected by Pacific Blue-conjugated NEO-201 antibody (BioLegend, San Diego, CA). To detect the NK markers modulated by ALT-803, purified NK cells were labeled with following antibodies: CD56-PE (clone 5.1H11), CD16-PerCP-Cy5.5 (clone 3G8), Tim-3-PE-Cy7 (clone F38–2E2), NKG2D-BV421 (clone 1D11), CD107a-APC-Cy7 (clone H4A3), Granzyme B-FITC (clone GB11), PD-1-APC (clone EH12.2H7), and CD158d-APC (clone mAb 33) (BioLegend). After staining, cells were washed twice with cold PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA). Analysis of cellular fluorescence was performed using BD FACSuite software (BD Biosciences). Positivity was determined by using fluorescence-minus-one controls.
7
Quantitative Analysis of NEO-201 Binding
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Binding of NEO-201 to human carcinoma cell lines was analyzed by flow cytometry. Cells (1.0 × 106) were incubated with 1 µL per test of LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA, USA) in 1× phosphate buffered saline (PBS) for 30 min at 4°C to accomplish live versus dead cell discrimination. Cells were then centrifuged, washed twice with cold PBS, and then stained with Pacific Blue-conjugated NEO-201 antibody (BioLegend, San Diego, CA, USA) in 1× PBS + 1% BSA (Teknova, Hollister, CA, USA) for 30 min at 4°C. After staining, cells were washed twice with cold PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). Analysis of cellular fluorescence was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). Positivity was determined using fluorescence minus one controls. Staining values >10% positive were considered positive for NEO-201 expression. Positive cell lines were ranked according to their quantified expression level (% positive × MFI), and then sorted into groups of low (<200), medium (200–1,000), and high (>1,000) expression.
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