To examine the contents of CD4 T-cells, a total of 1×106 splenocytes were stained with PerCP/Cy5.5-CD4 antibody (Ab) (Biolegend, San Diego, CA) and analyzed in BD FACSVerse (BD Bioscience, San Jose, CA). For the assessment of intracellular GRAIL expression, 1×106 cells were first stained with PerCP/Cy5.5-CD4 Ab and then fixed and permeabilized with IntraPrep (Beckman Coulter, Fullerton, CA), followed by reacting with rabbit-anti-GRAIL primary Ab (Abcam, Cambridge, MA), for 45 min. After washing, the cells were stained with FITC-anti-rabbit-IgG (Southern Biotech, Birmingham, AL) and then subjected to acquisition by BD FACSVerse. Data were analyzed by FlowJo software (Tree Star, Ashland, OR) with 20,000 events per sample. Isotype controls and Fc blocker (anti-mouse CD16/32; clone: 93; eBioscience, San Diego, CA) were used for the samples.
Intraprep
Manufactured by Beckman Coulter
Sourced in United States
The IntraPrep is a sample preparation product designed for the intracellular staining of cells. It is a reagent system used to facilitate the detection and analysis of intracellular antigens by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Cytomics fc500, by Beckman Coulter (2 mentions)
Gallios cytometer, by Beckman Coulter (2 mentions)
Facsverse, by BD (1 mentions)
Rabbit anti grail primary ab, by Abcam (1 mentions)
Anti mouse cd16 32 clone 93, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ, by Thermo Fisher Scientific (1 mentions)
Perm buffer 3, by BD (1 mentions)
Ebioscience foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Rotina 420r centrifuge, by Hettich (1 mentions)
U bottom plate, by BD (1 mentions)
Monensin, by Merck Group (1 mentions)
Brefeldin a, by BD (1 mentions)
Epics xl, by Beckman Coulter (1 mentions)
Facsflow, by BD (1 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
18 protocols using intraprep
1
Quantifying GRAIL Expression in CD4+ T-Cells
2
Characterizing T Cell Responses to Mycobacterial Antigens
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For determination of IFN-γ-producing cells, spleen CD4 T cells were stimulated with either PMA/Ionomycin or anti-CD3/CD28, and lung cell suspensions from M. tuberculosis-infected mice were incubated with either 5 μg/ml ESAT61-15, 5 μg/ml TB10.44-11 or PMA/Ionomycin for 6 h at 37o C. Brefeldin (10 μg/ml) was added to the cultures the last 4 h of stimulation. The IFN-γ secretion in naïve cells were then stained with live/dead staining followed by cell population-specific antibody cocktails. After labeling, cells were subsequently fixed and permeabilized using the leukocyte permeabilization reagent kit IntraPrep™ (Beckman, Brea, CA) and further stained with anti-IFN-γ (eBioscience, San Diego, CA). Data were acquired as described above.
To determine the expression of CTLA-4, Foxp3 and Ki-67, cell suspensions were prepared as described above were fixed and permeabilized using the eBioscience FOXP3/Transcription Factor Staining Buffer Set (Invitrogen) according to the manufacturer’s protocol and stained with specific antibodies for FOXP3, Ki-67 and CTLA-4.
To evaluate the expression of phospho-ribosomal protein S6, CD4 T cell suspensions were stained for surface markers as above, fixed with 2% PFA for 30 min on ice followed by permeabilization with Perm Buffer III (BD) for 30 min on ice.
To determine the expression of CTLA-4, Foxp3 and Ki-67, cell suspensions were prepared as described above were fixed and permeabilized using the eBioscience FOXP3/Transcription Factor Staining Buffer Set (Invitrogen) according to the manufacturer’s protocol and stained with specific antibodies for FOXP3, Ki-67 and CTLA-4.
To evaluate the expression of phospho-ribosomal protein S6, CD4 T cell suspensions were stained for surface markers as above, fixed with 2% PFA for 30 min on ice followed by permeabilization with Perm Buffer III (BD) for 30 min on ice.
3
Cell Surface and Intracellular Staining
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For cell surface markers, cell suspensions were incubated (20 min, 4°C) with the indicated fluorochrome‐labeled or biotinylated monoclonal primary antibodies in phosphate‐buffered saline with 1% BSA and 0.02% NaN3 (PBS staining buffer). For intracellular labeling, cells were fixed and permeabilized with IntraPrep (Beckman Coulter), followed by intracellular staining with indicated antibodies. Cells were analyzed on Cytomics FC500 or Gallios cytometers (both from Beckman Coulter) and data analyzed using FlowJo software.
4
Stem Cell Characterization by Flow Cytometry
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Stem cell characterization and differential expression at P2 and P3 were examined using flow cytometric analysis. Briefly, cells from P2 and P3 (early passages) were harvested using trypsin-EDTA digestion, centrifuged at 489 × g for 8 min at 4°C and re-suspended at a concentration of 106 cells/ml in DMEM/2% FBS. Aliquots containing 105 cells were incubated with individual surface antigen-specific fluorescent-labeled antibodies (Table II ) for 30 min at room temperature, and cells were then washed in PBS containing 2% FBS. For the analysis of the intracellular proteins nestin and SRY-box 2 (Sox2), permeabilization medium (Intra prep; Beckman Coulter, Inc., Brea, CA, USA) was used prior to the addition of the primary antibody. Finally, the cells were fixed in 10% formalin prepared in PBS containing 2% FBS, and analyzed using a flow cytometer (Beckman Coulter flow cytometry system with CXP software analysis; Beckman Coulter, Inc., Brea, CA, USA).
5
Quantifying Fibroblast αSMA Expression
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Each fibroblast subpopulation cultured for 7 days was centrifuged; the cells were fixed in 10% buffered formalin for 15 min at 25 °C, and then permeabilized for 5 min using IntraPrep (Beckman Coulter, Brea, CA, USA) according to the manufacturer’s instructions. After centrifuging and rinsing in FACS buffer, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-αSMA antibody for 30 min at 25 °C. As an isotype control, FITC-conjugated anti-mouse IgG2a (Thermo Fisher Scientific) was used. After centrifuging and rinsing in FACS buffer, the FITC fluorescence intensity of the cells was measured using the FACSAria system. Flow cytometry analysis was performed in triplicate using three independent samples.
6
Immunofluorescence and Flow Cytometry Staining
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Cells were stained with antibodies for 20 minutes on ice and expression markers were analyzed with a BD LSR II flow cytometer (BD Biosciences). For immunofluorescence staining, 1 × 105 cells in 200 μL/chamber were used, and for flow cytometry, 1 × 105 in 50 μL antibody were used. For intracellular staining, cells were fixed and permeabilized with IntraPrep (Beckman Coulter, Krefeld, Germany) according to the manufacturer’s protocol. For immunofluorescence staining, cells were centrifuged for 5 min at 200× g (4 °C) on culture slides using a Rotina 420R centrifuge (Hettich, Tuttlingen, Germany) and dried overnight. Cytospins were fixed with acetone:methanol (1:1) (AppliChem), blocked with 5% BSA (Sigma-Aldrich, Schnelldorf, Germany) in PBS and stained with primary and secondary antibodies (Table 1 ) diluted in 0.1% BSA in PBS. Cells were then fixed with 4% PFA (Microcos, Germany) and mounted with ProLong® Gold antifade reagent with DAPI (Invitrogen). The antibodies used in this study are listed in Table 1 . For analysis of dead cells, Propidium iodide (Sigma-Aldrich) was added for 10 min at 4 °C prior to the analysis by flow cytometry.
7
Quantifying T-cell Cytokine Responses via Flow Cytometry
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Freshly isolated T-cell fractions (effector cells) were rested in a 24 well plate in TCM overnight (1 × 107 cells per mL/well). On day 1, 5 × 105 effector cells/ 200 µL in RPMI were seeded in a 96 well U-bottom plate (BD Biosciences, Heidelberg, Germany) and stimulated with 1 µg/mL ppCMV_pp65 or ppCMV_IE1. As a positive control, cells were stimulated with 50 ng/mL PMA and 500 ng/mL Ionomycin and as negative controls; only culture medium and effector cells without stimulation were used. The plates were incubated at 5% CO2 and 37 °C for 60 min. Brefeldin A (1 μg/mL, BD Biosciences, Heidelberg, Germany) and/or Monensin (10 μg/mL, MerckKGaA, Darmstadt, Germany) was added and the cells were incubated for 16 h. Cells were then permeabilized with IntraPrep (Beckman Coulter, Brea, CA, USA), stained with respective antibodies and analyzed by flow cytometry. At least 30,000 events were acquired and gated based on the light scatter properties of lymphocytes followed by IFN-γ+CD3+, IFN-γ+CD8+, and IFN-γ+CD4+ T-cell populations.
8
Three-Color Flow Cytometry Analysis
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Three-color flow cytometric analyses were performed. Cells were stained with appropriate antibodies on ice, as previously described, for 20 minutes to detect cell surface markers. In some experiments, cells were further fixed and permeabilized with Intraprep (Beckman Coulter, Fullerton, CA, USA) and stained intracellularly with anti-TLR9. After washing, the cells were immediately subjected to flow cytometry (EPICS XL, Beckman Coulter, Tokyo, Japan), and analyzed using EXPO32TM software. Isotype-matched antibodies were used to determine the level of non-specific staining.
9
P-gp Expression Analysis by Flow Cytometry
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P-gp was studied by using UIC2 (Immunotech, France), monoclonal antibody, followed by labeling with a secondary antibody conjugated with phycoerythrin. Cells (1×106) were fixed and permeabilized using IntraPrep™ (Beckman Coulter, Villepinte, France) as per the manufacturer’s instructions. Fluorescence was measured and analyzed by flow cytometry. Protein expression for each transporter was quantified as the mean fluorescence intensity (MFI) shift (ratio of the MFI of antibody and isotype control). All experiments were performed in triplicate.
10
Phenotyping of MPE Cell Populations
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MPE samples were centrifuged at 500× g at 4 °C for 5 min to collect cells. Each aliquot of MPE cells was incubated with the mouse IgG anti-human monoclonal antibodies, as shown in Supplementary Table S2 . After incubation for 1 h at 4 °C, cells were washed with FACSFlowTM (BD Biosciences, Franklin Lakes, NJ, USA) and were examined for phenotyping. For intracellular staining of CD68, we used IntraPrep (Beckman Coulter, Inc., Miami, FL, USA) for fixation and permeabilization according to the manufacturer’s instructions.
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