To analyze the surface expression of GlyRα2, neutrophils (3 × 106/ml) were stimulated with E. coli for 5 min in RPMI-1640. Then, the neutrophils were fixed with Cytofix (BD Pharmingen) for 5 min. After washing with Pharmingen Stain buffer (bovine serum albumin, BSA) (BD Pharmingen), the neutrophils were stained with Alexa Fluor 488 carboxylic acid succinimidyl ester-conjugated anti-GlyRα2 for 30 min at room temperature. Anti-GlyRα2 antibody was conjugated with Alexa Fluor 488 carboxylic acid succinimidyl ester, in accordance with the manufacturer’s protocol. Neutrophils were washed in Pharmingen Stain buffer (BSA) (BD Pharmingen) and acquired on Guava Easycyte (GE Healthcare, Chicago, IL, USA). Analysis was performed using FCS Express V3 (De Novo Software, Los Angeles, CA, USA). Alexa 488-conjugated mouse IgG1 (BD Pharmingen) was used as an isotype control. To analyze the surface expression of flavocytochrome b558, neutrophils (3 × 106/ml) were stimulated with E. coli at a ratio of 1:10 for 20 min in RPMI-1640. Then, the neutrophils were washed with Pharmingen Stain buffer (BSA) (BD Pharmingen) and stained with PE-labeled anti-flavocytochrome b558 (MBL International, Woburn, MA, USA) for 1 h, and acquired on Guava Easycyte (GE Healthcare). Analysis was performed using FCS Express V3 (De Novo Software).
Pharmingen stain buffer bsa
Manufactured by BD
Sourced in United States
PharMingen Stain Buffer (BSA) is a laboratory reagent used for immunostaining procedures. It is a buffered solution containing bovine serum albumin (BSA) as the primary component. The buffer is designed to maintain the stability and activity of antibodies and other biomolecules during the staining process.
Automatically generated - may contain errors
Lab products found in correlation
Facs caliber flow cytometer, by BD (2 mentions)
Anti f4 80 fitc, by BioLegend (1 mentions)
Fitc annexinv solution, by BioLegend (1 mentions)
Trustain fcx anti mouse cd16 32 antibody, by BioLegend (1 mentions)
Facsdiva software, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Guava easycyte, by GE Healthcare (1 mentions)
Pharmingen stain buffer, by BD (1 mentions)
Fcs express v3, by De Novo Software (1 mentions)
Cytofix, by BD (1 mentions)
Edu click proliferation kit, by BD (1 mentions)
Fluorescence microscope, by Keyence (1 mentions)
Accutase, by BD (1 mentions)
Fitc conjugated cd133, by Miltenyi Biotec (1 mentions)
7 protocols using pharmingen stain buffer bsa
1
Analyzing Surface Expression of GlyRa2 and Flavocytochrome b558 in Neutrophils
2
Peripheral Blood CD11b+ Cell Analysis
3
Cell Phenotyping and Apoptosis Analysis
4
Peripheral Blood CD11b+ Cell Analysis
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RBC-lysed single-cell suspensions were obtained from peripheral blood and stained with fluorochrome-conjugated CD11b antibodies in PharMingen Stain Buffer (BSA) (BD PharMingen). Cells were analyzed on FACSCaliber flow cytometer (BD Biosciences) and analyzed with FlowJo software (Three Star).
5
Phenotypic Analysis of Peripheral Blood Cells
6
EdU Cell Proliferation Assay Protocol
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Six hundred forty-seven EdU Click Proliferation Kit (BD, San Jose, CA, USA) was used to assay the proliferation carefully following the manufacturer’s protocol. Briefly, cells were seeded in 96-well plates at 1000 cells per well in culture medium with 10 μM/ml EdU (n = 3 for each group). Cells were cultured for 7 days then washed with BD Pharmingen™ Stain Buffer (BSA) (BD, San Jose, CA, USA) and fixed with Fixative Solution (4% paraformaldehyde-based, component of 647 EdU Click Proliferation Kit); EdU in the cells was detected using a fluorescence microscope (Keyence, Itasca, IL, USA). EdU-positive Cell numbers were counted and calculated in same-size and random selected areas near the center of the original images of 3 experimental replicate wells using ImageJ (“analyze particles” function).
7
Identification of RCC Stem Cell Subpopulations
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Confluent cultures of the renal cell carcinoma (RCC) cell lines (HTB44, HTB47 and CRL1933) were washed two times with PBS and the cells were detached using Accutase (BD Biosciences) and centrifuged at 2000 rpm at 4 °C for 5 min. The cell pellet was washed once and re-suspended in BD Pharmingen™ stain buffer (BSA). Cells were diluted to 1 × 106 cells/mL with the stain buffer. For every 100 µL of cell suspension, 10 µL of fluorescein isothiocyanate (FITC) conjugated CD133 and/or phycoerythrin (PE) conjugated CD24 antibody (Miltenyi Biotec Inc., Gaithersburg, MD, USA) was added to the tubes. The cells were incubated for 30 min on ice in the dark and then washed two times with stain buffer and re-suspended in 1 mL of stain buffer. Cells were analyzed using SONY flow cytometer SP800S. The singlet population was gated as FSC vs. SSC, following which the selected population was graphed as PROM1 vs. CD24 plot. Four-quadrant data analysis was used to separate double positive vs. single positive cell populations. Based on the compensation measurements, the threshold for positive signal was set to ≤1 × 103 for both markers at the x and y-axis.
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