After measurements of AHR, the trachea was cannulated and the bronchial alveolar lavage (BAL) fluid was collected as described before39 (link). Briefly, we tracheostomized and intubated the mice and then washed the airways three times with 1 mL of ice-cold PBS each time, followed by centrifuging at 400 × g for 7 min and harvesting the cells. Data were analyzed with FlowJo software (TreeStar, Ashland, Ore). The absolute cell numbers in BAL fluid were calculated by means of flow cytometry by staining the cells with phycoerythrin (PE)–anti-Siglec-F (E50-2440; BD Biosciences, San Jose, Calif; 1/1000), fluorescein isothiocyanate(FITC)–anti-CD19 (6D5; 1/400), peridinin-chlorophyll-protein complex (PerCP)/Cy5.5–anti-CD3ε (17A2; 1/200), allophycocyanin (APC)–anti–Gr-1 (RB6-8C5; 1/1000), PE/Cy7–anti-CD45 (30-F11; 1/500), APC/Cy7–anti-CD11c (N418; BioLegend, San Diego, Calif; 1/400), and eFluor450–anti-CD11b (M1/70; eBioscience, San Diego, Calif; 1/500) in the presence of anti-mouse FC-block (2.4G2; BioXcell, West Lebanon, NH; 1/200). We used CountBright Absolute Count Beads (Thermo Fisher Scientific, Waltham, Mass), according to the manufacturer’s instructions. At least 105 CD45+ cells were acquired on a BD FACSCanto II (BD Biosciences) using the BD FACSDiva software v8.0.1.
Countbright absolute count beads
Manufactured by Thermo Fisher Scientific
CountBright™ Absolute Count Beads are a set of standardized polystyrene microbeads designed for cell counting applications. The beads are of a known concentration, allowing for the determination of the absolute count of cells or particles in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Ls column, by Miltenyi Biotec (3 mentions)
Quadromacs separator, by Miltenyi Biotec (3 mentions)
Anti pe microbeads, by Miltenyi Biotec (3 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (2 mentions)
Lympholyte mammal cell separation reagent, by Cedarlane (2 mentions)
M1 70, by Thermo Fisher Scientific (1 mentions)
Anti cd11c, by BioLegend (1 mentions)
Pe anti siglec f e50 2440, by BD (1 mentions)
Efluor 450, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Fixation permeablization buffer, by Thermo Fisher Scientific (1 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (1 mentions)
Facscalibur platform, by BD (1 mentions)
Lympholyte h, by Cedarlane (1 mentions)
Facsaria 3 cell sorter flow cytometer, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
6 protocols using countbright absolute count beads
1
Quantifying Airway Inflammatory Cells
2
Comprehensive Phenotyping of Immune Cell Subsets
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Cultured cells or cells harvested from peripheral blood were stained with fluorochrome-conjugated anti-rat CD4, CD25, CD80, CD86, CD11c, CD11b, IL-4, MHC-II, IFN-γ, and ROR-γt, Foxp3 Abs (BD Biosciences) and analyzed for expression of various cell surface or intracellular markers using a FACSCalibur platform (BD Biosciences). For intracellular cytokine (IL-4, IFN-γ) detection, cells were cultured with cell stimulation cocktail (protease inhibitor) for 16 h, then fixed and permeabilized with Fixation/Permeablization Buffer (eBioscience, MA) according to the manufacturer’s protocol. CD4+ T cell subsets were defined by expression of CD4 and IFN-γ for T helper (Th)1, expression of CD4 and IL-4 for Th2, expression of CD4 and ROR-γt for Th17 and expression of CD4, CD25, and Foxp3 for Treg. Cell apoptosis was tested using the Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences). For the enumeration of peripheral blood DC and CD4+ T cell subsets, CountBright absolute count beads (ThermoFisher Scientific, MA) were mixed with the cell samples and assayed via flow cytometry. Data were analyzed using FlowJo software (Tree Star, OR).
3
Flow Cytometric Enumeration of Mutant Erythrocytes
4
Multiparametric Flow Cytometry Analysis
5
Quantifying Cell Viability and Antigen Density
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Cell viability was determined by using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) following the manufacturer’s protocol. Cells that were negative for both propidium iodide and Annexin V expression were considered viable cells, while cells that were positive for Annexin V, individually or together with propidium iodide, were considered apoptotic cells.
Antigenic density was performed following the protocol developed by Lopez-Perez et al. (34 (link)). Briefly, the cells were washed with PBS and incubated with 2 µL of BV421 Mouse Anti-Human Alkaline Phosphatase (BD Biosciences) for 20 minutes. Then, the cells were fixed with 4% formaldehyde for 20 minutes. Finally, the cells were washed twice with PBS and resuspended in 100 µL. To standardize the expression of TNSALP variants on cell membranes, 5 µL of CountBright™ Absolute Count Beads (Invitrogen) were resuspended in 100 µL of PBS. All results were obtained with the BD FACSAria III Cell Sorter flow cytometer (BD Biosciences) on a logarithmic scale. Antigenic density was calculated as the ratio of the median intensities of the TNSALP-positive cells versus the median intensity obtained by the CountBrigh Absolute Count Beads.
Antigenic density was performed following the protocol developed by Lopez-Perez et al. (34 (link)). Briefly, the cells were washed with PBS and incubated with 2 µL of BV421 Mouse Anti-Human Alkaline Phosphatase (BD Biosciences) for 20 minutes. Then, the cells were fixed with 4% formaldehyde for 20 minutes. Finally, the cells were washed twice with PBS and resuspended in 100 µL. To standardize the expression of TNSALP variants on cell membranes, 5 µL of CountBright™ Absolute Count Beads (Invitrogen) were resuspended in 100 µL of PBS. All results were obtained with the BD FACSAria III Cell Sorter flow cytometer (BD Biosciences) on a logarithmic scale. Antigenic density was calculated as the ratio of the median intensities of the TNSALP-positive cells versus the median intensity obtained by the CountBrigh Absolute Count Beads.
6
Isolation and Analysis of Mouse Blood Cells
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PCH (CAS no. 36670‐1; purity ≥98%) was purchased from Sigma‐Aldrich, Oakville, ON, Canada. Lympholyte®‐Mammal cell separation reagent was purchased from CedarLane, Burlington, NC. Anti‐PE MicroBeads, LS Columns, and a QuadroMACS™ separator were from Miltenyi Biotec, Bergisch Gladbach, Germany. CountBright™ absolute count beads and fetal bovine serum (FBS) were purchased from Invitrogen, Carlsbad, CA. Anticoagulant solution, buffered salt solution, nucleic acid dye solution (contains SYTO® 13), anti‐CD24‐PE, and anti‐CD61‐PE were from Mouse Blood In Vivo MutaFlow® Kits (Litron Laboratories, Rochester, NY). Hank's balanced salt solution (HBSS) was purchased from MediaTech, Herndon, VA. Reagents used for flow cytometric micronucleus scoring (anticoagulant solution, buffered salt solution, stock propidium iodide solution, anti‐CD71‐FITC, and anti‐CD61‐PE solutions, stock RNase solution, and malaria biostandards) were from In Vivo Mouse MicroFlow® Kits (Litron Laboratories).
For the lacZ assay, phenyl‐beta‐D‐galactopyranoside (P‐Gal) was purchased from G‐Biosciences, St Louis, MO; proteinase K was from Invitrogen, Burlington, ON, Canada; packaging extract kits were from Agilent Technologies, Mississauga, ON, Canada; all other chemicals were purchased from Sigma‐Aldrich.
For the lacZ assay, phenyl‐beta‐D‐galactopyranoside (P‐Gal) was purchased from G‐Biosciences, St Louis, MO; proteinase K was from Invitrogen, Burlington, ON, Canada; packaging extract kits were from Agilent Technologies, Mississauga, ON, Canada; all other chemicals were purchased from Sigma‐Aldrich.
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