To enumerate the circulating hematopoietic stem cells, peripheral blood cells were incubated at 4°C with biotin-conjugated monoclonal Abs against lineage markers (CD3, CD11b, CD45R/B220, Ly-6C/G, TER119; Pharmingen 559971), PE-conjugated Sca-1 (Pharmingen 553336), followed by streptavidin-FITC (Pharmingen 554060). Cultured BMC were labeled with biotinylated anti-PDGFR-β ab (R&D system BAF1042), followed by streptavidin-FITC (Pharmingen 554060) or APC-conjugated anti-PDGFR-β ab (Biolegend 136008), PE/Cyanine7-conjugated anti-PDGFR-α ab (Biolegend 135911) or Cy3-conjugated α-SM actin ab (Sigma C6198). Cellular viability was assessed using trypan blue staining. The Sca-1+Lin− cells and PDGFR-β+ cells were counted using a FACS Sort (Becton Dickinson) or CytoFLEX Flow Cytometer (Beckman Coulter) and CellQuest software or Flow Jo software. The gating strategy analyses used are presented in Supplemental Figures I -IV .
Streptavidin fitc
Manufactured by BD
Sourced in United States
Streptavidin-FITC is a conjugate of streptavidin, a protein derived from the bacterium Streptomyces avidinii, and the fluorescent dye fluorescein isothiocyanate (FITC). This conjugate is commonly used in various laboratory applications that involve the detection and localization of biotinylated molecules.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin pe, by BD (3 mentions)
Pe cy5 labeled anti mouse cd11c clone n418 hamster igg, by BioLegend (2 mentions)
Fitc labeled anti mouse cd3ε clone 145 2c11 hamster igg, by BioLegend (2 mentions)
Fitc labeled anti mouse cd25 clone pc61 rat igg1λ, by BioLegend (2 mentions)
Pe labeled anti mouse cd8a clone 53 6.7 rat igg2aκ, by BioLegend (2 mentions)
Pe cy5 labeled anti mouse cd4 clone gk1.5 rat igg2bκ, by BioLegend (2 mentions)
Pe labeled anti mouse nk1.1 clone pk136 mouse igg2aκ, by BD (2 mentions)
Biotin fasl clone mfl3 armenian hamster igg2, by BD (2 mentions)
Annexin 5 fitc, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Epcam apc, by BioLegend (1 mentions)
Krt20, by Agilent Technologies (1 mentions)
Ncl l ki67 mm1, by Abcam (1 mentions)
Fluorescein isothiocyanate (fitc), by Jackson ImmunoResearch (1 mentions)
E cadherin, by Thermo Fisher Scientific (1 mentions)
P19arf, by Abcam (1 mentions)
H3k27me3, by Merck Group (1 mentions)
Alexa 488, by Jackson ImmunoResearch (1 mentions)
27 protocols using streptavidin fitc
1
Enumeration of Hematopoietic Stem Cells
2
Immunohistochemical Profiling of Skin Samples
3
Flow Cytometry Analysis of Immune Cells
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Antibodies used in the flow cytometry analysis were as follows: fluorescein isothiocyanate (FITC)-labeled anti-mouse Ly-6G/Ly-6C (Gr-1; clone RB6-8C5) rat IgG2bκ, phycoerythrin (PE)-labeled anti-mouse F4/80 (clone CIA3-1) rat IgG2bκ, PE/Cy5-labeled anti-mouse CD11c (clone N418) hamster IgG, FITC-labeled anti-mouse CD3ε (clone 145–2C11) hamster IgG, PE/Cy5-labeled anti-mouse CD45R/B220 (clone RA3-6B2) rat IgG2aκ, FITC-labeled anti-mouse CD25 (clone PC61) rat IgG1λ, PE-labeled anti-mouse CD8a (clone 53–6.7) rat IgG2aκ, and PE/Cy5-labeled anti-mouse CD4 (clone GK1.5) rat IgG2bκ were from BioLegend, Inc. (San Diego, CA, USA). PE-labeled anti-mouse NK1.1 (clone PK136) mouse IgG2aκ, biotin-FasL (clone MFL3) Armenian hamster IgG2, FITC-streptavidin and FITC-annexin V were from BD Pharmingen (San Diego, CA, USA). Monocytes/macrophages were defined as SSChiF4/80hiGr-1lo population, granulocytes as SSChiGr-1hi cells and dendritic cells as SSCloF4/80-cells. B cells, T cells, NK cells, NKT cells were defined as SSClo CD45R+, SSCloCD3ε+NK1.1-, SSCloCD3ε+NK1.1+, and SSCloCD3ε+NK1.1+, respectively. CD4 T cells (CD4+CD8-), CD8 T cells (CD4-CD8+), and regulatory T cells (CD4+CD8-CD25+) were also counted. Values represent the percentage of the non-parenchymal cells in each organ.
4
I-A(q) Expression Analysis in Cell Lines
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The expression of I-Aq on the NIH/3T3, thymic fibroblasts, and LS48 cells was analyzed by fluorescence microscopy using a monoclonal biotin mouse anti-mouse I-A(q) antibody (clone KH116, BD Biosciences, San Jose, CA, USA) and FITC Streptavidin (BD Biosciences, San Jose, CA, USA). Cells were seeded on coverslips in 12-well plates (1 × 105 cells/well) and cultured in a humidified incubator at 37 °C with 5% CO2 in Dulbecco’s Modified Eagle Medium, DMEM (1 mL/well) supplemented with 10% fetal calf serum (FCS) and antibiotics (100 IU/mL penicillin and 100 μg/mL streptomycin). After 24 h, the coverslips with the adhered cells were washed with phosphate buffered saline (PBS) and stained with KH116/FITC Streptavidin for 15 min at 37 °C in the dark. After washing three times with PBS, the cells were analyzed with a fluorescent microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a camera.
5
Quantifying Biotin-Streptavidin Binding on Erythrocytes
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To determine the substitution level of biotin molecules per erythrocyte, biotinylated erythrocytes and FITC-streptavidin (Nanjing Xinfan Biological Technology Co., Ltd, Nanjing, China) were incubated at 37 °C for 30 min. After removal of unattached FITC-streptavidin, the relative levels of NHS-biotin-streptavidin-FITC binding on erythrocytes were determined by flow cytometry (FCM) (BD FACSCalibur, USA) using FITC fluorescence.
To determine the efficiency of s-NPs anchored on biotinylated erythrocytes, biotinylated erythrocytes and FITC-tagged s-NPs were incubated at 37 °C for 30 min. After removal of unattached FITC-tagged s-NPs, the relative levels of FITC-tagged s-NP binding on biotinylated erythrocytes were determined by FCM using FITC fluorescence.
To determine the efficiency of s-NPs anchored on biotinylated erythrocytes, biotinylated erythrocytes and FITC-tagged s-NPs were incubated at 37 °C for 30 min. After removal of unattached FITC-tagged s-NPs, the relative levels of FITC-tagged s-NP binding on biotinylated erythrocytes were determined by FCM using FITC fluorescence.
6
Multicolor Flow Cytometry Analysis of Immune Cells
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The percentage of spleen cells was evaluated by flow cytometry (BD Biosciences, Oxford, UK) using the following antibodies: fluorescein isothiocyanate (FITC)-labeled anti-mouse Ly-6G/Ly-6C (Gr-1; clone RB6-8C5) rat IgG2bκ, phycoerythrin (PE)-labeled anti-mouse F4/80 (clone CIA3-1) rat IgG2bκ, PE/Cy5-labeled anti-mouse CD11c (clone N418) hamster IgG, FITC-labeled anti-mouse CD3ε (clone 145-2C11) hamster IgG, PE/Cy5-labeled anti-mouse CD45R/B220 (clone RA3-6B2) rat IgG2aκ, FITC-labeled anti-mouse CD25 (clone PC61) rat IgG1λ, PE-labeled anti-mouse CD8a (clone 53-6.7) rat IgG2aκ, and PE/Cy5-labeled anti-mouse CD4 (clone GK1.5) rat IgG2bκ, which were from BioLegend, Inc. (San Diego, CA). PE-labeled anti-mouse NK1.1 (clone PK136) mouse IgG2aκ, biotin-FasL (clone MFL3) Armenian hamster IgG2, FITC-streptavidin, and FITC-annexin V were from BD Pharmingen. Monocytes/macrophages were defined as SSChiF4/80hiGr-1lo population, granulocytes as SSChiGr-1hi cells, and dendritic cells as SSCloF4/80-cells. B cells, T cells, NK cells, and NKT cells were defined as SSClo CD45R+, SSCloCD3ε+NK1.1-, SSCloCD3ε+NK1.1+, and SSCloCD3ε+NK1.1+, respectively. CD4 T cells (CD4+CD8-), CD8 T cells (CD4-CD8+), and regulatory T cells (CD4+CD8-CD25+) were also counted. Values represent the percentage of the non-parenchymal cells in each organ.
7
Binding of Mannose-Binding Lectin to Natural Killer Cells
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For each sample, 2 × 105 purified NK cells were resuspended in Tris-buffered saline (TBS, pH 7.4), supplemented with 1% bovine serum albumin (BSA) and subsequently incubated with indicated concentration of bio-MBL or bio-HSA at 4°C for 1 h, followed by washing with TBS to remove the unbound proteins. Cells were then stained with streptavidin-FITC (BD Biosciences Pharmingen, San Diego, CA). The binding of MBL was analyzed by FACSCalibur (Becton Dickinson, Mountain View, CA, USA); cells stained with streptavidin-FITC served as the control. For competition studies, NK cells were preincubated with 50 μg/ml mannan, CLR, or CRD of MBL 4°C for 30 min.
For ELISA, 2 × 105 purified NK cells were pretreated with or without CLR of MBL at 4°C for 30 min, followed by incubation with indicated concentrations of MBL at 4°C for 1 h, then washed with TBS to remove the unbound proteins. Subsequently, the cells were coated onto ELISA wells (Nunc, Kamstrup, Denmark) by centrifugation and subsequently fixed with 4% paraformaldehyde for 15 min at RT. After that, the cells were washed with TBS and then incubated with mouse anti-MBL antibody (3B6) following by goat anti-mouse IgG H&L (HRP). The levels of bound MBL were determined using colorimetric assays.
For ELISA, 2 × 105 purified NK cells were pretreated with or without CLR of MBL at 4°C for 30 min, followed by incubation with indicated concentrations of MBL at 4°C for 1 h, then washed with TBS to remove the unbound proteins. Subsequently, the cells were coated onto ELISA wells (Nunc, Kamstrup, Denmark) by centrifugation and subsequently fixed with 4% paraformaldehyde for 15 min at RT. After that, the cells were washed with TBS and then incubated with mouse anti-MBL antibody (3B6) following by goat anti-mouse IgG H&L (HRP). The levels of bound MBL were determined using colorimetric assays.
8
Apoptosis Assay in Mouse Cells
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CL429 (Cat. Code: vac‐c429) was purchased from InvivoGen. Lipopolysaccharide (LPS) from Escherichia coli was purchased from Sigma (St.Louis, MO, USA), and Pam3CSK4 was obtained from EMC Microcollection (Tübingen, Germany). The TransDetect Annexin V‐FITC/PI Cell Apoptosis Detection Kit was purchased from TransGen Biotech (Beijing, China). RPMI 1640 and foetal bovine serum (FBS) were supplied by Gibco. Streptavidin FITC, anti–Mouse CD117 APC eFluor 780, and anti‐Mouse Ly‐6A/E (Sca‐1) PerCP‐Cyanine5.5 were purchased from BD Pharmingen (San Diego, CA). Lineage Cell Detection Cocktail‐Biotin was purchased from Miltenyi Biotec (Germany). Lipofectamine 3000 was purchased from Thermo Fisher. Antibodies were purchased from Affinity Biosciences (Jiangsu, China) and Cell Signaling Technology (Massachusetts, USA). Commercial ELISA kits were purchased from DAKEWE (Beijing, China).
9
Flow Cytometric Characterization of Adherent and Suspension Cells
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Adherent cells were washed with Dulbecco's PBS (DPBS) (Thermo Fisher, Waltham, MA, #12563011) and treated with Tryp‐LE Select (Thermo Fisher, Waltham, MA, #12563011) at 37°C for 5 minutes, followed by gentle pipetting to complete cell dissociation. The dissociated cells were then washed with DPBS several times before antibody staining for flow cytometry. For suspension hematopoietic cells, gentle pipetting was used to dislodge any loosely adhered hematopoietic cells from the adherent stromal layer, and the cells were washed multiple times with PBS before staining. Cells were stained in ice‐cold fluorescence‐activated cell sorting buffer (PBS, 1% BSA). Antibodies used; KDR‐PE (BD Pharmingen, San Jose, CA, #560872), CD34‐APC (BD, San Jose, CA, #555824), CD144‐PE (BD, San Jose, CA, #561714), CD45‐PE (BD, San Jose, CA, #561866), CD117‐APC (Thermo Fisher, Waltham, MA, clone 104D2), CD117‐PE (BD, San Jose, CA, #555714), CD235a‐FITC/PE (BD, San Jose, CA, Clone GA‐R2 (HIR2)), CD41a‐APC (BD, San Jose, CA, #559777), CD71‐FITC (BD, San Jose, CA, #555536), CD73‐PE (BD, San Jose, CA, #561014), CXCR4‐Biotin (BD, San Jose, CA, #555973), and Streptavidin‐FITC (BD, San Jose, CA, #554060). Flow cytometry was conducted on a Stratadigm S1000EXI or Beckton Dickinson (BD) dual‐laser FacsCalibur flow cytometer. Analyses were conducted using FlowJo v8.7 (FlowJo, LLC) software.
10
Hematopoietic Cell Phenotypic Analysis
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Hematopoietic cell phenotypic analysis was completed by flow cytometry. Briefly, 5 × 106 hematopoietic cells harvested from mouse bone marrow were blocked with Fc-block for 10 min, incubated with biotin-conjugated lineage antibodies (CD4, CD8, CD45R/B220, Gr-1, Mac-1 and Ter-119) and then stained with streptavidin-FITC (BD Pharmingen), c-Kit-APC (BD Pharmingen), and Sca1-PE (eBioscience). The frequencies of HPCs (Lin-Sca1-c-kit1 + cells), LSK cells (Lin-Sca1 + c-kit + cells), and HSCs (Lin-Sca1 + c-kit + CD150 + CD48-cells) were analyzed with a flow cytometer (LSRII flow cytometer). Data were analyzed using FlowJo software.
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