Briefly, BrdU (500μl of 10mg/ml stock) was injected IP into pregnant mice at E16.5 30 min before tissue harvest. Two dissected retinas (3 replicates for WT, 4 replicates for CKO) per tube were trypsinized, fixed in ethanol and permeabilized with 2N HCl/0.5% Triton X-100 for 30 min. After blocking with 1% BSA in PBS, cells were stained with BrdU antibody overnight at 4°C. After 3 rounds of washing with PBS containing 1% BSA, cells were stained with Alexa488 conjugated goat anti-rat secondary antibody (Life Tech.). Antibody-labeled cells were treated with RNase (0.25μl of 20mg/ml) and propidium iodide (10μl of 1mg/ml) before undergoing flow cytometry analysis using Guava EasyCyte (Millipore). Cell cycle fraction analysis was done with GuavaExpressPro (Millipore). Age–matched C57BL/6 (Taconic) embryos were used as WT controls due to the difficulties in obtaining multiple WT or CKO from the same litter.
Guavaexpresspro
Manufactured by Merck Group
Sourced in Germany
The GuavaExpressPro is a compact flow cytometry system designed for cell analysis and counting. It provides accurate and reliable results for a variety of applications, including cell viability, apoptosis, and cell cycle analysis. The system features intuitive software, automated setup, and easy-to-use features for efficient cell analysis.
Automatically generated - may contain errors
Lab products found in correlation
Guava easycyte, by Merck Group (1 mentions)
C57bl 6, by Taconic Biosciences (1 mentions)
Alexa fluor 488 conjugated antibody, by Thermo Fisher Scientific (1 mentions)
Guava easycyte plus flow cytometer, by Merck Group (1 mentions)
Anti human cd45, by BioLegend (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Guava easycyte 5, by Merck Group (1 mentions)
Pe conjugated mouse igg1, by BioLegend (1 mentions)
Fitc conjugated mouse igg1, by BioLegend (1 mentions)
Guava easycyte 6ht, by Merck Group (1 mentions)
4 protocols using guavaexpresspro
1
BrdU-Labeling for Cell Cycle Analysis in Retina
2
CAIX Expression Quantification in Hypoxic Cells
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After 48 h of hypoxic incubation, transiently transfected cells were scraped into culture medium, centrifuged at low speed, washed twice with Versene solution and incubated with the CAIX-specific mouse monoclonal M75 antibody (1 µg/mL, BMC SAS, Bratislava, Slovakia) for 30 min at 4 °C. Following the centrifugation and washing with Versene, cells were incubated with the secondary anti-mouse Alexa Fluor 488-conjugated antibody (Invitrogen, Carlsbad, California) diluted 1:1000 in 1% BSA for 30 min at 4 °C. Pelleted cells were again washed twice with Versene and finally analyzed using a Guava easyCyte plus flow cytometer (Millipore, Darmstadt, Germany). Data were analyzed with Cytosoft 5.2 software using Guava Express Pro (Millipore, Darmstadt, Germany). Debris, cell doublets and clumps were excluded from analyses by scatter gating, and a total of 10,000 single cells were analyzed for each sample.
3
Characterization of Mesenchymal Stem Cells
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MBMSCs were analyzed by flow cytometry to detect cell surface antigens previously reported as criteria for identifying MSCs [23 (link)]. MBMSCs were harvested with trypsin and EDTA, centrifuged at 1500×g for five minutes, and resuspended at 1 × 104 cells/ml in PBS containing 0.5% bovine serum albumin, 2 mM EDTA, and FcR Blocking Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany). Aliquots were then incubated with individual antibodies: anti-human CD73 (phycoerythrin (PE)-conjugated mouse IgG1; BioLegend, San Diego, CA, USA), anti-human CD90 (fluorescein isothiocyanate (FITC)-conjugated mouse IgG1; BioLegend), anti-human CD105 (FITC-conjugated mouse IgG1; Ancell Corp., Bayport, MN, USA), anti-human CD14 (FITC-conjugated mouse IgG1; BioLegend), anti-human CD34 (PE-conjugated mouse IgG1; BioLegend), and anti-human CD45 (FITC-conjugated mouse IgG1; BioLegend). FITC-conjugated mouse IgG1 (BioLegend), and PE-conjugated mouse IgG1 (BioLegend) were the isotype controls. Flow cytometric analysis was performed with Guava easyCyte 5 (Merck, Darmstadt, Germany), and data analysis was performed using Guava Express Pro (Merck).
4
Quantifying Circulating Cell Populations
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To assess the composition of the circulating cell population, samples (n = 4 per time point) were analyzed with a benchtop flow cytometer (Guava easyCyte 6HT; Merck Millipore). Samples were centrifuged at 200 g and resuspended in PBS. Debris was excluded using a threshold on the forward scatter signal and at least 20,000 events were counted per sample. Data analysis was performed using Guava Express Pro software (Guava Express Pro; Merck Millipore) in combination with InCyte software (InCyte v2.7; Merck Millipore). Granulocytes were separated from the agranulocytes by gating first on cell size and granularity (forward scatter and side scatter, respectively) and on color in a second gating step. Data were normalized to the amount of circulating cells at the start of the experiment. The best fit was determined for either a line or one-phase decay.
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