BIOMAG

Image Source: BD Biosciences, BD Influx Cell Sorter Waste Tank : >Disconnect the Vacuum Pump and Fluidic Lines. >Open Waste Tank and Add 1% Bleach and discard. >Close the lid tightly (To ensure vacuum build up). >Reconnect the vacuum Pump and Fluidics Lines. Sheath Tank : >Release pressure using pressure knob. >Disconnect all the fluid lines connected to the sheath Tank. >Open and check the sheath level (if required, add sheath fluid to the sheath tank). >Close and connect the air and fluid lines. > Connect Sheath Line to Filter to Instrument. Switching ON : >Switch on Condenser, Vacuum Pump, Mains of the Instrument and Computer. >Wait for around 10 minutes to build the pressure in the waste tank and sheath tank. >Wait for 10 minutes for lasers to fully warm up and turn them ON one by one. >Make Sure, the displayed sheath pressure matches the pressure build scale on the sheath tank. >Attach the nozzle to the nozzle asse...

Fluorescence Microscopy and Flow Cytometry, both are biological instruments which are capable of measuring the biophysical and biochemical properties of cells or particles which are under study. Both of them are capable of identifying types of cells present in a given sample. These two technologies are useful in variety of applications and they have widespread usage... But, These two differ in their applications and use.  Fluorescence Microscopy Flow Cytometry Sample mounted on a glass slide. Sample is allowed to pass through a liquid stream through a sensing area. (Single cell suspension). Produces magnified images of the cells Does not produce the images. Whole Tissue section can be visualized and analyzed Tissue must be disintegrated into single cell suspension. And each single cell is analyzed. Detection through human eye and images can be produced on a computer screen Detection is...

Please Watch The Demo Video attached after the protocol and example Data!!!! Preparation of Samples for Cell Cycle using Flow Cytometry :  1) Suspend approximately million cells (10^6) in 1mL of 1x PBS (phosphate-buffered saline solution). and with the help of pipette, gently aspirate (up and down) about 7-8 times, to make the single cell suspension.  2) Take a centrifuge tube and add 4mL of 70% ethanol.. (70% ethanol should be in ice before using). and add 1mL of cells in PBS to the same tube. Keep the tubes on ice for minimum 2 hours. This step is to fix the cells. (Cells can be kept at 4℃ for a week). 3) Centrifuge the ethanol fixed cells for 5 minutes at 300g. Carefully take out the supernatant. Do not disturb the pellet.  4) Add 5mL of cold 1xPBS to this pellet and suspend the cells using pipette.  5) Centrifuge the tubes at 300g for 5 minutes. Discard the supernatant. Again do not disturb the pellet.  6) If required, ...

Immunophenotyping is a most important and regularly used technique  in Immunology and Immunological research studies. The technique is usually used for the disease monitoring and diagnostic purpose either by immunohistochemistry or by utilizing the high-throughput technique Flow Cytometry.. Flow Cytometry is a common method for the immuno phenotyping the different cell subsets or sub populations of cells. Immunophenotyping is used for the identification and characterisation of the cell surface markers and intracellular markers. In simple terms, the technique is used to detect and study the proteins expressed by the cells. Samples used for the study could be Tissue Section (Fresh or Fixed) or Cell suspension (mono-dispersed cell suspension for FCM). This technique helps in rapid and easy phenotyping of each cell in a heterogeneous sample according to the presence or absence of a protein combination. Markers (or proteins of interest) are usually functional membrane proteins...

Flow Cytometry Analysis using Peripheral Blood Cells: 1) Collect approximately 75 microliters of blood into 1mL PBS containing 5microM EDTA and mix immediately to prevent clotting. keep the tubes on ice. (10 microliters of 0.5M EDTA stock). 2) Remove Red blood cells from samples. [RBCs are lysed using either Gey's solution or a buffered ammonium chloride solution. (Or, BD sells a product called FACS lysis buffer" that is used after the staining protocol to lyse RBCs and fix the cells)]. Use proper solution according to the instructions given by the manufacturer.  3) Wash the cells at least 2-3 times with FACS buffer. (FACS buffer contains PBS solution supplemented with either 1% BSA or 5% FBS and 0.05% NaN3).  4) Suspend the pellet from the final wash in 50microliters FACS buffer per each analysis on a single sample. (Up to three separate staining reactions can be set up from a single sample).   5) Add 50 microliters of cell suspension to 10 micro...

Measuring Cells vs Measuring Cells in Suspension microscope vs flow cytometer                              F luorescence Microscopy and Flow Cytometry, both are biological instruments which are capable of measuring the biophysical and biochemical properties of cells or particles which are under study. Both of them are capable of identifying types of cells present in a given sample. These two technologies are useful in variety of applications and they have widespread usage.. But, These two differ in their applications .  Fluorescence Microscopy:   Microscope was invented before flow cytometry, fluorescence microscope is improved version of a light microscope, uses the lasers as excitation sources to excite the fluorescence present in the specimen under study. Fluorescence emitted from the cells or particles present in specimen produces a magnified image. Fluorescence microscope is useful t...

                        Flow Cytometry is a technique of identifying and measuring the physical and chemical properties of cells which travel in a suspension. The benefit of this technique is that it allows the rapid and simultaneous measurement of several parameters on a cell by cell basis. It can detect and analyze thousands of cells per second.   There are wide range of Applications of flow cytometry, some of them are explained below.  1) Immunological Applications :   Immunophenotyping (I t involves identification and characterization of cell surface proteins (CD markers) or intracellular proteins using fluorescent-conjugated antibodies. For example: identification of CD markers present in blood cells.)     Stem Cells Analysi s ( involves studying of stem cells such embryonic stem cells (ESCs), human induced pluripotent stem cells (hiPSCs), studying the pluripotency and multipotency...

                       The method of separation of cells from tissue or separation of cell sub populations from a heterogeneous population of cells is called cell sorting. Cell separation is commonly achieved by separating them based on cell size and cell shape, cell density, granularity and bio-marker expression or protein marker expression. There are various methods for cell sorting such as Density Gradient Centrifugation, Filtration, Aptamer binding, plastic/adhesion, LCM, RCB rosetting, FACS and MACS.  Commonly used are Fluorescence Activated Cell Sorting (FACS) and Magnetic Activated Cell Sorting (MACS).                  Fluorescence Activated Cell Sorting can be defined as the physical separation of sub populations of cells based on the protein expression markers which are tagged with fluorescence molecules, and subsequent collection of these sub populations in ...

                                 The speed of the flow cytometry offers wide range of data points and data plots. Due to its sensitivity and versatility, it has been used extensively. Flow cytometer can detect up to 1000s of cells per second.. But, it should be noted that the flow cytometry also involves the statistics, its significance, calculating the fluorescence intensity..                Statistics in flow cytometry involves, total number of data points acquired, percentage population, most importantly mean and median fluorescence intensity, and others. Most of us get confused about the term "MFI", whether it means mean fluorescence intensity or median fluorescence intensity.. Truth is, both.. It means it can be described as either mean or median fluorescence intensity. Some researchers use Mean for MFI and some use Median for MFI values.  (Keep...

                      The term "Compensation" in flow cytometers refers to the process of correcting fluorescence spill over. The compensation matrix ensures that the fluorescence emitted is detected in a particular detector which is from the fluorescence being measured. As we all know that most of the fluorescence molecules have broad emission range. if more than one one fluorescence is selected, their emission spectra may overlap. To correct this spectral overlap in cytometry data, a mathematical matrix called compensation is used. The Intention of this article is to teach you the compensation from basic theory to practical. This example for FITC and PE fluorescence molecules shows that, Some of the FITC fluorescence is being detected in PE Detector. and Some of the PE fluorescence is being detected in FITC Detectors. Task here is to make sure that in cytometry data, FITC is in FITC detector only and PE is in PE Detector only....

                Spectra Viewers are the fluorochrome tools which help in understanding the wavelength ranges of excitation and emission of fluorochromes.. These tools are very helpful in determining specific laser, detectors and range of fluorescence spectra. When Multi-Color Flow Cytometry is the experiment, then these must be used to analyze emission spectrum, fluorescence spill over and compensation values. How these tools help.. First, Lets consider a simple example of analyzing the spectrum for Green Fluorescence Protein (GFP) and you want to know the excitation wavelength and emission wavelength range for GFP. By knowledge, you know that GFP will excite with 488nm Blue laser and emits the green fluorescence at apporximately 510nm.. Now, just open a spectrum viewer and select the GFP as shown below. Now, you have analyzed GFP fluorescence and received the knowledge that It excites with 488nm Blue Laser and emission maximum is...