Questions tagged [flow-cytometry]
The flow-cytometry tag has no usage guidance.
35 questions
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CD45 as a microglial marker in Fluorescence-Activated Cell Sorting (FACS)
I am reading this article: Microglial cannabinoid receptor type 1 mediates social memory deficits in mice produced by adolescent THC exposure and 16p11.2 duplication.
In Figure 1a, the authors are ...
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Concentrating cell concentration after subsampling
I need some help with regards to a concentration correction. I am measuring how many cells detach from a rock vs sonication time. Becuase I start from a rock, I cannot make aliquots and measure ...
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How to correct the different (background) fluorescence for different cell types?
I am differentiating mouse bone marrow macrophages in vitro. However, I found that the differentiated macrophages have higher background fluorescence (the unstained sample) in all channels, which ...
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How can I easily distinguish between eukaryotic and prokaryotic cells using flow cytometry?
I am trying to count populations of cells in a co-culture. One organism is Pichia pastoris and the other organism is a gram-negative bacterium. Pichia stains gram-positive using crystal violet and the ...
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Fixing the cell with 4%PFA and staining with PI to observe cell cycle?
I am wondering whether we can replace 70% ethanol to fix and perm the cells with 4% PFA to observe the cell cycle. Do I understand correctly that 4% PFA can permeate the cells, thus the PI will ...
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Number of membrane proteins of a particular type per cell
Is it possible (or meaningful) to count how many proteins (protein copy number?) of a certain type a given cell has on its surface?
For instance, say there is some membrane integral protein ...
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How do I cover Cellstrainers?
I‘m doing some experiments for my bachelors thesis and I‘m using some Cell strainers by Roth for it (https://www.carlroth.com/com/en/accessories/cell-strainer-easystrainer™/p/cly9.1). My problem is ...
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Using Height and Area for plotting histogram of each marker in FACS
I had a flow cytometry experiment. After normal filter with cell debris and doublet cells, I would like to gate the cells by different markers. However, I get confused to use which parameter to plot ...
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Why am I finding peculiar B cell population when pbmcs are cultured for 3 days with CD40L?
While performing intracellular cytokine assay on B cells, I am finding a peculiar population.
I use 2*10^6 cells for studying each functional marker. The pbmcs are cultured for 72 hours, with CD40L ...
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Can I use fluorophore conjugated antibody as neutralizing antibody?
I need to order a neutralizing antibody to block a protein of interest.
The pure grade functioning antibody (no conjugates) is substantially more expensive than the fluorophore conjugatated ones.
Can ...
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CRISPR-Cas9 knockout
With CRISPR-Cas9 I have conducted a targeted knockout of a DNA region encoding a certain protein (working with leukocytes). My question is, how long does it take until this protein is not detectable ...
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Relationship between bacteria size and temperature?
I'm, doing a research in a TOC (total organic carbon) degradation in a BAC (biologically active carbon) filter for the grey-water treatment. Recently, I started to wonder if there is a relation ...
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DRAQ5 and PI incompatible?
I'm interested to analyse hemolymph cells from a marine mollusc. I wanna count only "alive" cells so my strategy was the following: DRAQ5+ (nucleated cells) and PI- (alive cells) and identify ...
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How are single cells sorted in an scRNA-Seq protocol by FACS?
I am computational guy trying to understand how FACS sorting works in an scRNA-Seq protocol. About sorting single cells in each well of a 384-well plate, I have a question that would be grateful if ...
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Non-nucleated cell-like population with RNA
We're working on invertebrate hemolymph (blood) and we have found with flow cytometry (staining with DRAQ5) a cell-like population without nucleus but it has RNA production.
Does anyone any ...
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Can flow-FISH be used on metaphase cells?
I know flow-FISH can be used on interphase cells but was wondering if it only works on interphase cells or can work on cells regardless of stage in the cell cycle.
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Flow cytometric counting of apoptotic adhering cells
I have been trying to measure rates of apoptosis in epithelial cells by marking apoptotic cells with annexin V conjugated with phycoerythrin (PE) and sorting using flow cytometry. Unfortunately, the ...
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Flow Cytometry Channels
I did a flow cytometry experiment for the first time. I had three conditions: (1) unstained healthy monocytes (2) healthy monocytes stained with green/red (488/570) from the Live/Dead thermofisher kit ...
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Is it possible to sort droplets inside an oil phase with a FACS
In a nutshell, is it possible to FACS a sample that is in oil rather than in a buffer (water based solution)?
I would greatly appreciate any help with this.
Thank you
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What enzymes I shall use for preparing single cell suspension from cardiac tissue for flow cytometry?
Can anyone suggest which enzymes I shall use for preparing single cell suspension from cardiac tissue. I will be looking at both cell surface and intracellular antigens involving cardiomyocytes and ...
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Flow Cytometry to learn about Cell cycle
I am posting my Flow Cytometry data here again because I realize that there are many people who are willing to help me, and I can learn a lot from you guys.
In this cell culture Lab, we use Cell U-...
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Flow cytometry data Analysis [closed]
everyone.
Since I am new in the flow cytometry technique, I am not really sure how to analyze the result.
We use cell U-937 Lymphoblast.All Cells
Could Anyone help to explain me this one?
Thanks ...
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Cell dye in red fluorescence spectrum
There are some cell dyes available for stianing cells intracellularly / membrane. I am aware of other channels (CFSE \ PKH26). But I want to stain cells for flow cytometry with a 'red' dye.
I have ...
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Flow cytometry analysis
I've performed an experiment wherein I have stimulated cells (cell line) with a drug across different time-points - in its unmodified (Drug) or modified form (H-Drug or M-Drug).
All cells are seeded ...
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Purpose of antibody wash
It is common practice after surface staining cells for flow cytometry analysis to wash the antibody out of solution before analyzing a sample. I have tried analysis with and without washing the ...
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Differing Flow Cytometry Genome Sizes and Sexual Mating Implies Differing Chromosome Counts
Do the fundamental principles of genetics strongly suggest the following is a derived conjecture?
If 2 flowering plant species A and B have widely differing genome size (at a minimum a 50% difference)...
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Staining cells for FACS at 4 degrees or ambient temperature
I'm sorting a 293 derived line. One thing that is worrisome for me is cell viability after the sort. Usually I have been staining and washing at room temperature (on the benchtop) on a nutator. I have ...
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Can flow cytometric analysis differentiate between a parent contributing longer chromosomes versus multiple smaller chromosomes?
For flowering plants, can flow cytometry of seed from muti-generational interaction of two species and their offspring help us decide if wide differences in genome size for the two species and their ...
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Jurkat single cell clonal expansion
I'm hoping to do a clonal expansion of Jurkat, expressing my construct which is currently transduced virally. From the FACS data I currently have roughly 30-40% expression in my culture but I need it ...
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Magnetic-activated cell sorting vs. FACS
When sorting cell populations it is possible to use either magnetic-activated cell sorting or fluorescence-activated cell-sorting (FACS). I am wondering when you would choose either technique and what ...
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Alternatives to commercially available flow cytometric analysis software for use in cell cycle analysis?
I know that many labs use either FlowJo or FCS Express to plot and analyze their flow data. These seem like the two standards for most labs (and I have used FlowJo before). However, my current funding ...
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Accuracy of genome size estimation by flow cytometry
I'm working on a genome project and using an in silico k-mer analysis to estimate the size of our genome based on the available Illumina reads. The k-mer based estimate is consistent across a wide ...
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Flow cytometry issues
I'm having problems with data analysis here.
I have flow cytometry data being collected on a Fortessa, and when I import them into FlowJo 8.7, all of my fluorescence values are systematically 10X ...
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Isotype control antibodies in Flow Cytometry
In a Flow Cytrometry, one can add an Isotype Control Antibody to allow another antibody to bind more specific to the cells.
My question is, how can the Isotype Control Antibody add specificity to the ...
Jeffrey
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Hoechst or DAPI for nuclear staining?
When is it best to use Hoechst vs. DAPI for nuclear staining? They seem to be very similar on paper. Are there situations where one is clearly preferable?
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