Brains were dissected in a cold Ringer’s solution, incubated with collagenase/dispase mix at 29°C (Roche, 15 minutes for larval and pupal brains and 30 minutes for adult brains), washed in dissociation solution (Sigma-Aldrich), mechanically dissociated into single cells and transferred via 35um mesh (Falcon) to eliminate clusters and debris. 1000 DsRed+ positive cells were sorted using a 100μm nozzle and low pressure in BD FACSAria Fusion (BD Bioscience) cell sorted directly into 100μl Pico-Pure RNA isolation kit extraction buffer (Life Technologies) followed by RNA extraction using the kit or stored in -80 for later use. To minimize the effect of an injury response, the samples were kept on ice for the entire procedure from dissection up to the RNA extraction except from the dissociation enzyme incubation.
Dissociation solution
Manufactured by Merck Group
Sourced in United States
Dissociation solution is a laboratory reagent used to facilitate the separation of cells or tissues into individual components. It contains a combination of enzymes and other compounds that disrupt the intercellular connections, allowing for the release and isolation of individual cells or cellular components. The core function of this product is to provide a standardized and effective means of cell dissociation for various applications in cell biology, tissue engineering, and biomedical research.
Automatically generated - may contain errors
Lab products found in correlation
Pico pure rna isolation kit extraction buffer, by Thermo Fisher Scientific (2 mentions)
Facsaria fusion, by BD (1 mentions)
Facscan, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Facsaria 3, by BD (1 mentions)
Collagenase dispase, by Roche (1 mentions)
Facscalibur machine, by BD (1 mentions)
Isotype matched irrelevant monoclonal antibodies, by BD (1 mentions)
Facsaria, by BD (1 mentions)
Alexa fluor 488 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Trypan blue, by Welgene (1 mentions)
Facscalibur, by BD (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Fitc conjugated anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Fitc conjugated anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
7 protocols using dissociation solution
1
Single-Cell RNA Isolation from Drosophila Brains
2
Comparative Analysis of AFP Promoter Variants
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The fluorescence-activated cell sorting (FACS) analysis was conducted to assess the specificity and transcriptional activity of modified AFP promoters with HREs located upstream. Cells were transduced with dAd/CMV-GFP, dAd/AFPm-GFP, dAd/a2bm-GFP, dAd/a2bSm-GFP, dAd/Ha2bm-GFP, or dAd/Ha2bSm-GFP (Huh7 and A549 at 40 MOI, HepG2 at 30 MOI, Hep3B and Hep1 at 20 MOI, and BJ at 100 MOI). At 48 h post transduction, cells were harvested using a dissociation solution (Sigma, St. Louis, MO) and washed with phosphate buffered saline (PBS). GFP expression levels from each group were analyzed by FACScan (Beckton-Dickinson, East Rutherford, NJ). Data were collected from 10,000 cells and analyzed with the CellQuest software (BD Biosciences Immunocytometry Systems, San Jose, CA).
To compare the promoter strength of full length hAFP promoter and a2bm promoter, Hep3B and Huh7 cells were transduced with replication-incompetent Ad expressing GFP under the control of hAFP promoter (dAd/hAFP-GFP) or dAd/a2bm-GFP at 40 or 50 MOI, respectively. GFP expression levels from each group were analyzed by FACScan (Beckton-Dickinson) as described above.
To compare the promoter strength of full length hAFP promoter and a2bm promoter, Hep3B and Huh7 cells were transduced with replication-incompetent Ad expressing GFP under the control of hAFP promoter (dAd/hAFP-GFP) or dAd/a2bm-GFP at 40 or 50 MOI, respectively. GFP expression levels from each group were analyzed by FACScan (Beckton-Dickinson) as described above.
3
Cell Adhesion Assay Protocol
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Cell adhesion assays were performed as described previously [20 (link)]. Briefly, collagen type I and FN (10 ng/mL, BD BioSciences, San Jose, CA) prepared in PBS containing 1 mM CaCl2 and 1 mM MgCl2 were coated on 96-well plates. As a control, wells were coated with 1% BSA overnight at 4°C. After being blocked with 1% BSA, 5 x 104 cells were removed by dissociation solution (Sigma), washed in serum-free medium, added to each well, and the cells were allowed to adhere to the plate for 45 min at 37°C. The plate was washed with PBS containing 1 mM CaCl2 and 1 mM MgCl2 until no cells remained in the wells coated with BSA. Cell adhesion was evaluated by staining adherent cells with 0.1% crystal violet, solubilizing cells with 0.2% Triton X-100 in PBS, and reading the absorbance at 630 nm with a Microplate Reader (Bio-Rad). All samples were done in triplicate and at least three different isolations of cells were used.
4
Astrocyte Isolation from Drosophila Brains
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Brain dissociation and sorting of astrocytes was done as described previously.31 (link) Briefly, L3 or adult brains were dissected in ice-cold Ringer’s solution, incubated at 29°C with collagenase/dispase (Roche, 15 min for larval brains and 30 min for adult brains), washed in dissociation solution (Sigma-Aldrich), mechanically dissociated by pipetting into single cells and transferred via a 35μm mesh (Falcon) to eliminate clusters and debris. Pools of 1000 dsRed+ positive cells were sorted using a 100μm nozzle and low pressure in BD FACSAria III (BD Bioscience) cell sorter directly into 100μL Pico-Pure RNA isolation kit extraction buffer (Life Technologies), followed by RNA extraction using the kit or stored in −80°C for later use. To minimize injury or stress responses, samples were kept on ice whenever possible.
5
Characterization of Cell Surface Markers
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Isolated cells were harvested using dissociation solution (Sigma, USA), washed twice with PBS and then stained with fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD45, CD34 and CD14 (BD Biosciences, USA) and phycoerythrin (PE)-conjugated mouse anti-human CD44 and CD166 (BD Biosciences, USA) and APC-conjugated mouse anti-human CD73. Isotype-matched irrelevant monoclonal antibodies (BD-Pharmingen, USA) were used as negative controls. After 30 minutes incubation at room temperature and washing with PBS, approximately 10,000 events were collected on a FACS Calibur machine (BD Biosciences, USA) using the Cell quest as data acquisition software and analyzed using FlowJo7.6 software for the graphical presentation of data.
6
Isolation and Clonogenic Assay of SP1-B7-Positive Cells
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A549 cells were dissociated by dissociation solution (Millipore) and filtered through 40 μm strainer and centrifuged at 1500 rpm for 3 min. Thirty-four million cells were incubated with 70 μg of SP1-B7 for 30 min at RT and washed by PBS (pH 7.4) including 5% FBS and further incubated with Alexa fluor 488-conjugated anti-mouse IgG (Life technologies) for 30 min at RT in the dark. After washes, the cells were isolated by BD FACSAria (BD Biosciences). After cell sorting, SP1-B7-positive or -negative cells were counted with 0.4% Trypan blue (Welgene) and live cells were used for the clonogenic survival assay.
7
Flow Cytometric Analysis of Cell Surface Markers
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Flow cytometric analysis was performed as described previously [14 (link)]. Briefly, cells were treated with dissociation solution (Millipore) and suspended in PBA (1% bovine serum albumin, 0.02% NaN3 in phosphate buffered saline (PBS), pH 7.4). Cells were then incubated with primary antibodies for 30 min at 4°C and further incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Santa Cruz Biotechnologies, Santa Cruz, USA) or FITC-conjugated anti-rabbit IgG (Santa Cruz Biotechnologies). After washing with PBA, propidium iodide (PI)-negative cells were analyzed for the antibody binding using FACSCalibur (BD Biosciences). For the analysis of annexin V-positive and csBAP31-positive A549 cells, A549 cells were first stained with SP1-B7 and phycoerythrin (PE)-conjugated anti-mouse IgG (Life technologies). Cells were further stained with annexin V-FITC (BD Biosciences) and PI before analysis. The paired t-test was a statistical procedure used to compare the difference between the mean fluorescence intensities (MFI) of two populations, and a p-value of less than 0.05 was considered statistically significant.
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