Flow cytometry was performed to analyze the cell-surface expression of typical protein markers. The adherent cells were incubated with the following anti-human primary antibodies CD31-phycoerythrin (PE), CD45-fluorescein isothiocyanate (FITC), CD90-R-PE, HLA-DR-R-PE (Becton-Dickinson, Franklin Lakes, NJ, USA). The total of 10,000 labeled cells were analyzed using a Guava easyCyte flow cytometer running Guava Express Plus software (Guava Technologies, Inc., Hayward, CA, USA).
Guava express plus software
Manufactured by Merck Group
Sourced in United States
The Guava Express Plus software is a flow cytometry analysis tool designed for use with Merck's Guava flow cytometry instruments. The software provides automated data analysis capabilities and supports multiple file formats for data import and export.
Automatically generated - may contain errors
Lab products found in correlation
Guava easycyte, by Merck Group (3 mentions)
Cd90 r pe, by BD (2 mentions)
Hla dr r pe, by BD (2 mentions)
Guava easycyte flow cytometer, by Merck Group (2 mentions)
Lipofectaminetm 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Eclipse ti s, by Nikon (2 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde solution, by Merck Group (1 mentions)
Anti mouse pe secondary antibody, by Merck Group (1 mentions)
Guava easycyte ht system, by Merck Group (1 mentions)
6 protocols using guava express plus software
1
Cell Surface Marker Analysis by Flow Cytometry
2
Cell Surface Marker Expression Analysis
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To analyze the cell-surface expression of typical protein markers, adherent cells were passaged with 0.25% trypsin (Gibco). The cells were washed in PBS and fixed in a 4% paraformaldehyde solution (both from Sigma-Aldrich, Milwaukee, WI, USA) for 10 min; and incubated with the following anti-human primary antibodies: mouse monoclonal CD45-phycoerythrin (PE) (Abcam, Cambridge, MA, USA; catalog no.: ab25603; dilution: 0.2 µg/106 cells), mouse monoclonal CD31-fluorescein isothiocyanate (FITC) (Abcam; catalog no.: ab33858; dilution:10 µl/106 cells), mouse monoclonal CD90-FITC (Abcam; catalog no.: ab11155; dilution: 10 µl/106 cells), mouse monoclonal HLA-DR-PE (Abcam; catalog no.: ab95830; dilution: 5 µl/106 cells). A total of 10,000 labeled cells were analyzed using a Guava easyCyte flow cytometer running Guava Express Plus software (Guava Technologies, Inc., Chicago, IL, USA).
3
Analysis of Cell Surface Markers
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To analyze the cell-surface expression of typical protein markers, adherent cells were incubated with the following anti-human primary antibodies: CD31-phycoerythrin (PE), CD45-fluorescein isothiocyanate (FITC), CD90-R-PE, HLA-DR-R-PE (Becton–Dickinson and Company, Franklin Lakes, NJ). Unconjugated markers were reacted with anti-mouse PE secondary antibody (Guava Technologies, Hayward, CA). A total of 10,000 labeled cells were analyzed using a Guava EasyCyte flow cytometer running Guava ExpressPlus software (Guava Technologies).
4
Multiparametric Flow Cytometry Analysis
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Subconfluent cells were collected with trypsin–EDTA and fixed with 4% PFA for 20 min at room temperature. After washing with PBS, the fixed cells were labeled with fluorescein isothiocyanate-conjugated antibodies against human CD14 (clone M5E2), CD90 (clone 5E10), and CD105 (clone 266) (Becton Dickinson), CD34 (clone 581) and CD44 (clone J.173) (Beckman Coulter, Fullerton, CA, USA), STRO-1 (clone STRO-1; Santa Cruz Biotechnology, Dallas, TX, USA), and phycoerythrin-conjugated mouse immunoglobulin (Ig) G1 (clone MOPC-21; Becton Dickinson). Flow-cytometric analysis was performed using a Guava™ EasyCyte HT system and Guava™ Express Plus software (version 2.7; Merck KGaA).
5
Endogenous Promoter Transcriptional Activity Evaluation
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CHO-K1, BHK-21 and HEK293T cells were transfected with 1 µg of novel vectors containing the endogenous promoters. The pCMV-ZsGreen1-1 was used as control to compare the transcriptional activity of endogenous promoters. Co-transfection of each plasmid with pCIneo-CMV-IFNα vector [29] was performed to evaluate transfection efficiency, determined by quantification of interferon alpha (IFNα) in culture supernatants. At the same time, novel vectors were co-transfected with pBKS plasmid used as a carrier to equalize DNA load across all transfections. Lipofectamine TM 2000 Transfection Reagent (Invitrogen) was used, according to the manufacturer's instructions.
After 48 h, transfection efficiency was analyzed by assessing fluorescence intensity of ZsGreen1 in transfected cells by fluorescence microscopy (Eclipse Ti-S, Nikon Instruments Inc) and flow cytometry (Guava EasyCyte, Merck Millipore). The percentage of ZsGreen1-expressing cells (%ZsGreen) and ZsGreen1 mean fluorescence intensity (MFI) of each sample were analyzed using Guava ExpressPlus software (Merck Millipore) and MFI was expressed as fluorescence arbitrary units (FAU). Additionally, the culture supernatant of pCIneo-CMV-IFNα co-transfected cells was collected and analyzed by ELISA sandwich using the protocol described by Ceaglio et al. (2008). [29]
After 48 h, transfection efficiency was analyzed by assessing fluorescence intensity of ZsGreen1 in transfected cells by fluorescence microscopy (Eclipse Ti-S, Nikon Instruments Inc) and flow cytometry (Guava EasyCyte, Merck Millipore). The percentage of ZsGreen1-expressing cells (%ZsGreen) and ZsGreen1 mean fluorescence intensity (MFI) of each sample were analyzed using Guava ExpressPlus software (Merck Millipore) and MFI was expressed as fluorescence arbitrary units (FAU). Additionally, the culture supernatant of pCIneo-CMV-IFNα co-transfected cells was collected and analyzed by ELISA sandwich using the protocol described by Ceaglio et al. (2008). [29]
6
Endogenous Promoter Transcriptional Activity Evaluation
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CHO-K1, BHK-21 and HEK293T cells were transfected with 1 µg of novel vectors containing the endogenous promoters. The pCMV-ZsGreen1-1 was used as control to compare the transcriptional activity of endogenous promoters. Co-transfection of each plasmid with pCIneo-CMV-IFNα vector [29] was performed to evaluate transfection efficiency, determined by quantification of interferon alpha (IFNα) in culture supernatants. At the same time, novel vectors were co-transfected with pBKS plasmid used as a carrier to equalize DNA load across all transfections. Lipofectamine TM 2000 Transfection Reagent (Invitrogen) was used, according to the manufacturer's instructions.
After 48 h, transfection efficiency was analyzed by assessing fluorescence intensity of ZsGreen1 in transfected cells by fluorescence microscopy (Eclipse Ti-S, Nikon Instruments Inc) and flow cytometry (Guava EasyCyte, Merck Millipore). The percentage of ZsGreen1-expressing cells (%ZsGreen) and ZsGreen1 mean fluorescence intensity (MFI) of each sample were analyzed using Guava ExpressPlus software (Merck Millipore) and MFI was expressed as fluorescence arbitrary units (FAU). Additionally, the culture supernatant of pCIneo-CMV-IFNα co-transfected cells was collected and analyzed by ELISA sandwich using the protocol described by Ceaglio et al. (2008). [29]
After 48 h, transfection efficiency was analyzed by assessing fluorescence intensity of ZsGreen1 in transfected cells by fluorescence microscopy (Eclipse Ti-S, Nikon Instruments Inc) and flow cytometry (Guava EasyCyte, Merck Millipore). The percentage of ZsGreen1-expressing cells (%ZsGreen) and ZsGreen1 mean fluorescence intensity (MFI) of each sample were analyzed using Guava ExpressPlus software (Merck Millipore) and MFI was expressed as fluorescence arbitrary units (FAU). Additionally, the culture supernatant of pCIneo-CMV-IFNα co-transfected cells was collected and analyzed by ELISA sandwich using the protocol described by Ceaglio et al. (2008). [29]
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