The expression of mHsp70 was quantified by flow cytometry using a BD FACSCalibur™ (BD Biosciences, Heidelberg, Germany). Briefly, harvested and washed cells (2x105) were incubated with FITC-cmHsp70.1 monoclonal antibody (mAb; 40 µg/mL, multimmune GmbH) in flow cytometry buffer (PBS containing 10% v/v FBS) for 30 min on ice (in the dark), after which the cell pellet was washed twice and re-suspended in flow cytometry buffer containing propidium iodide viability stain (PI; 1 μg/mL, Sigma) prior to analysis. At least 2x104 viable cells were acquired for each sample, from which the percentage of positive cells was determined. An isotype-matched mAb (mouse IgG1 FITC; BD Biosciences) was used as the control to define the mHsp70-positive region. The mean fluorescence intensity (MFI) was reported by subtracting the MFI of an isotype-matched control antibody from the MFI of cmHsp70.1 positively stained cells. The Quantibrite™ PE Phycoerythrin Fluorescence beads (BD, 340495) were used to quantify the number of Hsp70 molecules on the membrane per cell by setting a calibration curve relating to the MFI values. Data were analyzed using FlowJo™ software (version 10.1).
Mouse igg1 fitc
Manufactured by BD
Sourced in United States
Mouse IgG1 FITC is a fluorescently labeled antibody used in various laboratory applications. It is a primary antibody that binds to the IgG1 isotype of mouse immunoglobulins and is conjugated with the FITC (fluorescein isothiocyanate) fluorescent dye.
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Lab products found in correlation
Pe mouse igg1, by BD (6 mentions)
Facscalibur flow cytometer, by BD (4 mentions)
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Cd45 fitc, by BD (3 mentions)
Cd90 fitc, by BD (3 mentions)
Cd34 fitc, by BD (3 mentions)
Facscan, by BD (3 mentions)
Mouse igg2a pe, by BD (3 mentions)
Apc mouse igg1, by BD (2 mentions)
Cd90 pe, by BD (2 mentions)
Cd44 fitc, by BD (2 mentions)
Cd29 fitc, by BD (2 mentions)
Fitc mouse anti human cd4, by BD (2 mentions)
Facsflow, by BD (2 mentions)
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Annexin 5 fitc, by BD (1 mentions)
Anti cd19 apc, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Annexinv apc, by Beckman Coulter (1 mentions)
Mouse igg1, by Thermo Fisher Scientific (1 mentions)
29 protocols using mouse igg1 fitc
1
Quantification of Membrane-bound Hsp70 by Flow Cytometry
2
Flow Cytometry Analysis of T/B Cells and ZAP-70 Expression
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For extracellular staining of T and B cells, peripheral mononuclear cells were stained with annexin V-FITC, anti-CD3-PE, 7AAD, and anti-CD19-APC (BD Biosciences). Cells gated on either CD19+ or CD3+ were then analyzed for expressions of annexin V and 7AAD.
For intracellular staining of ZAP-70 in cell death experiments, RosetteSep and ficoll-purified CLL cells were first surfaced stained with annexin V-APC and 7AAD, then fixed with solution A (Beckman Coulter, Brea, CA, USA) for 12 min at 37 °C, washed with PBS, permeabilized with solution B (Beckman Coulter) for 5 min at room temperature, and then stained with mouse anti-human ZAP-70-FITC (Beckman Coulter) for 15 min. Mouse IgG1-FITC (BD) was used as an isotype control. Unstained and single-stained controls were always included in all flow cytometry experiments. All sample data were acquired on BD FACSCalibur and analyzed using CellQuest Pro software (BD). CLL cell samples were considered ZAP-70+ if ≥20% of the cells stained positively.
The ZAP-70 status of each patient sample was determined by diagnostic flow cytometry. Whole blood was stained with anti-CD19, anti-CD5, anti-CD38, and anti-ZAP-70. The negative control is a normal blood sample that should not have CD19+CD5+ leukemic cells. The positive control is the autologous CD19-CD5+ZAP-70+ T cells.
For intracellular staining of ZAP-70 in cell death experiments, RosetteSep and ficoll-purified CLL cells were first surfaced stained with annexin V-APC and 7AAD, then fixed with solution A (Beckman Coulter, Brea, CA, USA) for 12 min at 37 °C, washed with PBS, permeabilized with solution B (Beckman Coulter) for 5 min at room temperature, and then stained with mouse anti-human ZAP-70-FITC (Beckman Coulter) for 15 min. Mouse IgG1-FITC (BD) was used as an isotype control. Unstained and single-stained controls were always included in all flow cytometry experiments. All sample data were acquired on BD FACSCalibur and analyzed using CellQuest Pro software (BD). CLL cell samples were considered ZAP-70+ if ≥20% of the cells stained positively.
The ZAP-70 status of each patient sample was determined by diagnostic flow cytometry. Whole blood was stained with anti-CD19, anti-CD5, anti-CD38, and anti-ZAP-70. The negative control is a normal blood sample that should not have CD19+CD5+ leukemic cells. The positive control is the autologous CD19-CD5+ZAP-70+ T cells.
3
Cell Surface Marker Analysis by Flow Cytometry
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For cell-surface molecular analysis, cells were suspended in PBS containing 1% FBS, and then stained with PE-conjugated anti-CD86 (12-0869-42), FITC-conjugated anti-CD206 (MA5-16870), PE-conjugated anti-CD163 (12-1639-42), Mouse IgG2b κ Isotype Control PE (12-4732-81), Mouse IgG1, κ Isotype Control Alexa Fluor 488 (53–4714) (all the antibodies purchased from eBioscience, USA), PE/cyanine 5-conjugated anti-CD11b (E-AB-F1081G, Elabscience, China), PE/cyanine 5 Rat IgG2b, κ Isotype Control (E-AB-F09842G, Elabscience, China), CD4-FITC/CD8-PE/CD3-PerCP (340298), CD3-FITC/CD16+56 PE (340042), Mouse IgG1 PE (349043), Mouse IgG1 FITC (349041), Mouse IgG2a PerCP (349054) (all the antibodies purchased from BD, USA) for 30 min at 4°C. For flow cytometry (FCM) gating, cells were stained with isotype-matched control antibodies or unstained cells and other cells were analyzed according to that fating strategy. Specimens were subsequently analyzed by FCM (Navios, Beckman).
4
Cell Surface HSP70 Expression Analysis
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LS174T and UD‐5 cells (2 × 105 cells) were suspended in flow cytometry buffer containing either FITC‐labeled cmHsp70.1 mAb (20 µg mL−1, multimmune GmbH) or respective isotype control (mouse IgG1 FITC; BD Biosciences) for 30 min on ice in the dark. Cells were washed twice, followed by re‐suspension in a flow cytometry buffer with 1 µg mL−1 of PI.[61 (link)
] Samples were acquired by flow cytometry and analyzed using FlowJo software.
] Samples were acquired by flow cytometry and analyzed using FlowJo software.
5
Characterization of Osteoblast Phenotype
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The OBs were suspended in Dulbecco’s phosphate-buffered saline (PBS) and incubated with antibodies against mouse IgG1-PE,mouse IgG1-APC, mouse IgG1-FITC and mouse IgG1-PerCP (BD, Franklin Lakes, NJ, USA) as a negative control. Antibodies against CCR1-PE,CD138-FITC,CD45-APC and CD34-PerCP (BD) were used to stain the experimental sample. After incubation in the dark at 4°C for 30 min,the cells were washed twice with PBS. At least 5 × 104 cells were acquired and analyzed using a FACSCalibur flow cytometer (BD Biosciences). OBs are marked as CD138−CD45−CD34− cells.
6
Stem Cell Surface Marker Profiling
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Cell surface markers specific for stem cells were assessed by flow cytometry, including CD73, CD90, CD105, CD166, CD14, CD34, CD45, HLA-ABC, and HLA-DR [12 (link)]. Cells from P3–P5 were collected and resuspended in PBS at a concentration of 2 × 104 cells/20 μl. Then, they were incubated with the following antibodies for 20 min at RT in the dark: mouse IgG1-PE, mouse IgG1-FITC, CD90-FITC, CD105-FITC, CD73-FITC, CD14-PE, CD34-FITC, CD45-FITC, HLA-ABC-FITC, and HLA-DR-PE (BD Biosciences, San Jose, USA). Then, the cells were washed with PBS and centrifuged at 800 rpm for 5 min. After the supernatant was discarded, the cells were resuspended with 300 μl of PBS. The antibody-labelled cells were analysed with a BD FACSCalibur flow cytometer. The data were analysed using CellQuest Pro software, which was provided by the manufacturer.
7
Flow Cytometric Characterization of Adherent Cells
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At day 3 or 7 of differentiation, adherent cells were harvested using 0.25% TrypleE with EDTA and made into a single-cell suspension in PBS with 0.2% BSA; then all cells were analyzed using flow cytometry directly without purification. Mouse Anti-Human APJ APC-conjugated Antibody (R&D, Catalog Number: FAB8561A) was used as a ratio of 1：50, Mouse IgG1 (FITC, Material Number: 551954, BD Pharmingen), Mouse Anti-human CD31 (CD31-FITC, Material Number: 555824, BD Pharmingen), Mouse IgG1-APC (Order no: 130092214, Miltenyi Biotec), Mouse Anti-human CD34 (CD34-APC, Material Number: 560940, BD Pharmingen), Mouse Anti-human CD43 (CD43-APC, Material Number: 560198, BD Pharmingen), Mouse Anti-Hamster IgG PE-conjugated Antibody (Catalog Number: F0120, R&D system), Mouse Anti-human KDR (KDR-PE, FAB357P, R&D) and Mouse Anti-Human NRP-1 (NRP-1-PE, Material Number: 565951, BD) antibodies were used at a ratio of 1：20. Single-cell suspensions were subse-quently incubated with antibody or antibodies at 4℃ for about 40 mins. Flow cytometric detection of the cell surface antigens were performed on a BD AccuriTM C6 Plus personal flow cytometer (Becton Dickinson). Compensation was set by single-positive controls.
8
Quantifying FcγR Expression in THP-1 Cells
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The THP‐1 cell line (ATCC) was grown in RPMI culture medium at 37 °C and 5% CO2 atmosphere. The cells were harvested by centrifugation (300 RCF, 5 min) and concentrated to 2 × 106 cells/mL in PBS with 2.5% BSA and 0.1% sodium azide (FACS buffer). To determine FcγRI expression, the cells were incubated with 10 μg/mL mouse anti‐human FcγRI antiserum (R&D) for 1 h on ice. Mouse IgG1 (Sigma) was used as an isotype control. The cells were washed twice with the FACS buffer by centrifugation at 300 RCF for 5 min and incubated with 1 : 100 FITC‐conjugated anti‐mouse immunoglobulins antibody (The Binding Site). For FcγRII and FcγRIII expression, the cells were incubated with 0.25 μg/test of FcγRII‐FITC or FcγRIII‐FITC antibody (eBioscience) for 1 h on ice. Mouse IgG1‐FITC (BD Biosciences) was used as an isotype control. The cells were washed twice, and fluorescence intensity was measured by BD FACSCanto™ II Flow Cytometer (BD Biosciences) using BD FACSDiva™ software. The data were analysed using FlowJo V10.1 software.
9
Membrane Hsp70 Expression Profiling
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The membrane Hsp70 (mHsp70) phenotype was analyzed by flow cytometry using the FITC-conjugated cmHsp70.1 monoclonal antibody (mAb, IgG1, multimmune GmbH, Munich, Germany) on a FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany). Tumor cells (0.2 x 106 cells) were washed with flow cytometry buffer (PBS/10% v/v fetal bovine serum, FBS) and incubated either with the cmHsp70.1 mAb or with an isotype matched FITC-labeled control immunoglobulin (mouse IgG1 FITC, 345815, BD Biosciences) on ice in the dark for 30 min. After a second washing step, viable cells (propidium iodide negative cells) were gated upon, and the proportion of positively stained cells were analyzed.
10
Intracellular Cytokine Profiling of T Cells
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For intracellular cytokine staining for PBMC or CD4+ T cells, assays were carried out with staining buffers and antibodies from BD Biosciences. Briefly, cells were seeded into a 96-well plate (up to 1 × 106 cell per well) and stimulated with PMA (100 ng/ml) and ionomycin (1 μg/ml) in the presence of GolgiStop for 4 h. After stimulation, cells were fixed with BD Cytofix fixation buffer and washed with BD Perm/Wash buffer. Cells in each well were equally divided into two wells, with one for intracellular cytokine staining and the other for isotype control staining. The following fluorophore-conjugated antibodies from BD Biosciences were used for staining analysis or as isotype controls: anti-CD4-pacific blue (clone: RPA-T4; 1:330), anti-IL-17A-Alexa647 (clone: N49-653; 1:20), anti-IFN-γ-FITC (clone: B27: 1:100), anti-IL-10-PE (clone: JES3-19F: 1:660), mouse IgG1-Alexa647 (clone: MOPC-21; 1:40), mouse IgG1-FITC (clone: MOPC-21; 1:100), and rat IgG2a-PE (clone: R35-95: 1:660). Stained cells were analyzed with a BD LSR II cytometer. Cytokine secretion in CD4+ lymphocytes was accessed with FlowJo.
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