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细胞分选平台
    平台简介应用&服务平台成员招聘信息

Introduction

The cell sorting platform of SIAIS serves as a technical and scientific resource by providing flow cytometric cell analyzing and cell sorting for Shanghaitech University and the surrounding biotech and biomedical research communities. This essential resource aides performing researches in antibody drug discovery, immunology, cancer and stem cell biology etc.

Flow cytometry is a highly sensitive technology, which can quantitatively measure fluorescence emitted from cells and physically isolate target cells at single-cell level. By using specific fluorescent probes, this technology allows simultaneous multi-parametric analysis and sorting of tens of thousands of cells per second, and enable investigators to rapidly analyze complex samples and enrich desired cell subsets with high purity. It is an indispensable and powerful tool for studies of biology, biomedicine and related fields.

The cell sorting platform is equipped with advanced instruments, which include BD FACSAriaIII, BC Cytoflex S, BC Moflo Astrios EQ, BD Influx, Amnis ImageStream MarkII, Miltenyi AutoMACS Pro. And excellent physical conditions were also designed for instruments operating. We will establish SOPs (Standard Operating Procedures) for routine work procedures to ensure the integrity, quality, consistency, reproducibility of data or outcomes under the guide of flow cytometry shared resource laboratory (SRL) best practices.

Instruments

● BD FACSAriaIII
BD FACSAriaIII is a highly automated digital flow cytometer. Also as a quartz cuvette flow cell based benchtop cell sorter, it has excellent sensitivity. The automatic sterilization fluidics, fixed-alignment optical system and perfect automatic quality control program make it easy to operate. Because of its easy-to-use, BD FACSAria series had popularized the complex cell sorting to a broad audience of researchers.

Specification:
    Lasers: 488nm, 561nm, 633nm, 407nm/375nm;
    13 fluorescence detectors, 1 FSC, 1 SSC;
    4-way sorting, plate sorting up to 384 well;
    ADC 18 bit 10MHZ;
    Nozzle: 70/85/100/130 (μm)
    Detector Configuration

Detector Configuration



BD Influx

The innovative nozzle component allows high droplet frequencies at relatively low sheath pressures, enabling high-speed sorting while maintaining best cell viability. Itsmodular architecture requires full hands-on controls and provides configurable detecting channels to meet application needs and easily exchangeable fluidics from sample line to sheath line.


Specification:

    Lasers: 355nm, 445nm, 488nm, 561nm, 640nm;
    20 fluorescence detecting channel, 1 SSC (488nm);
    2 SPO (Small Particle Option) FSC, size resolution > 100nm;
    Support up to 6-way sorting;
    Plate sorting up to 384 well, single cell index sorting enabled;
    ADC 16bit 40MHZ;8 Pulse processors
    Nozzle size: 70/86/100/140/200 (μm) for selection






● BC Moflo Astrios EQ
A reliable high speed cell sorting system with the best comprehensive sorting performance. Its sophisticated design and manufacturing ensure a high stability of the jet. The most powerful electronic system is an important guarantee to achieve high purity, high recovery and high speed simultaneously. The automated drop delay and monioring functhion, IntelliSort II, work well with 70μm and 100μm nozzle. With FSC enhancement module, this instrument can resolve minimum 0.1μm diameter particles and so its application range has been extended further. Its unique function of uncharged droplets sorting provides an ultra-high speed and great recovery pre-enrichment of very rare cells.

Specification:
    Lasers: 355nm (elliptical focus),
               405nm (flat-top focus),
               488nm (flat-top focus),
               532nm (flat-top focus),
               561nm (flat-top focus),
               592nm (flat-top focus),
               642nm (flat-top focus);
    28 fluorescence detecting channels, 1 SSC(488nm), 1 SSC(405nm);
    FSC enhancement module: 2 FSC PMT channels;
    Support up to 6-way sorting;
    Plate sorting up to 1536 well, index sorting;
    32bits for pulse height parameters with 100MHZ sampling;
    Integrated BSL-2 biosafety cabinet;
    Nozzle size: 50/70/80/100/120/150/200 (μm)


● Amnis ImageStream MarkII
A quantitative imaging flow cytometry analyzer. By electronically tracking and photographing stably moving cells in sheath flow, this instrument combines the speed, sensitivity, and phenotyping abilities of flow cytometry with the detailed imagery and functional insights of microscopy. Besides fluorescence intensities, this combination provides, more importantly, single cell based statistical analysis of a number of morphological parameters such as Area, Major axis, Minor axis, Circularity, Similarity, Aspect Ratio, Spot count, and others. Flow speed and auto-focus monitoring design maintains good imaging quality.

Specification:
    Laser: 488nm (200mW), 561nm (200mW), 405nm(120mW), 642nm(150mW);
    2 TDI CCD provide 12 imaging channels;
    Objective lenses: MultiMag 60X (resolution 0.33μm per pixel, 0.9NA),
    40X (0.5μm per pixel, 0.75NA),
    20X (1μm per pixel, 0.5NA)
    785nm(70mW) SSC (in channel 6 or 12)
    Bright field in all channels for selection, colored LED illumination.

Featured Applications
    1)Cell signaling. NF-κB translocation to nucleus, HIV induced NFAT, Foxp3 localization.
    2Internalization and phagocytosis. CpGB internalization, phagocytosis of bacteria by monocytes.
    3Surface and intracellular co-localization. Ligand colocalization to lysosmes
    4Cell shape change and chemotaxis. MCP-1 activation of monocytes differentiation of FGCP cells
    5Cell-cell interaction. Immune synapse formation, NF-κB activation from T-cell APC conjugation
    6Cell cycle and mitosis. Morphological classification of mitosis without specific fluorescent probes.
    7Cell death and autophagy. Apoptosis, nuclear fragmentation, Caspase 3 activation.
    8Stem cell biology. Eryithroid differentiation
    9Microbial cells observation. Bacterial phagocytosis in PBMC

Spectral Imaging Channels And Corresponding Fluorophores


● BC Cytoflex S
A compact benchtop analyzer with the highest sensitivity. In this instrument, a 1.3 NA objective lens integrated with quartz cuvette flow chamber allows more photons being collected, and FADP detectors are used instead of PMTs to acquire much higher photon-electron conversing efficiency. Those innovative designs together with other improvements in optics and fluidics bring a breakthrough sensitivity. Its alignment free optics, auto-cleansing fluidics, auto QC program and friendly software interface make it very easy to use and maintenance.

Specification:
    Laser: 405nm (80mW), 488nm(50mW),561nm(50mW),638nm(50mW);
    13 fluorescent channels, 1 FSC, 1 SSC(488nm);
    96-well plate loader

Detector Configuration


● Miltenyi AutoMACS Pro

The AutoMACS Pro separator is a benchtop automated magnetic cell sorter. The most advantage of it is the capability of batched gentle isolation of massive cells in a very short time. Applicable samples of it include many cell types from multiple species, Such as T/B/NK/DC/Treg/monocytes in peripheral blood, bone marrow and spleen, yeast cells, circulating tumor cells, CD34+ cells, CD133 cells , several kinds of neuron cells and so on. Magnetic cell separation is particularly useful for pre-enriching rare cell populations prior to flow cytometry cell sorting. And this conjunction strategy results in better purity and shorter working time.

Specification:
    Capacity of AutoMACS columns: 105--4X109 cells per sample
    Cell binding capacity: 2X109 magnetic bead labeling cells per sample
    Sequentially processing 6 samples at a time
    Cell suspension volume: 0.25-50mL, or 0.25-15mL whole blood
    Programmable operating procedures, Automatic sampling and elution;
    Capable of operating in biosafety cabinet


General protocol for flow cytometry cell analyzing and sorting

The procedure of any flow cytometry experiment may be divided into four steps: cell samples acquisition, cells staining with fluorescence, cells detecting and/or sorting on flow cytometer, data analysis and biological messages deciphering. We describe here a detailed common protocol with key notes as following.

1. Acquire single healthy cells, which in general are in middle term of exponential growth or from fresh tissues. Of course, cells preserved by other method are also acceptable.
(Note: Usually to prepare enough cells is quite important.)

2. Handle cells according to the experiment scheme. Usually make into treated and untreated groups.

3. Divide those cell samples into control group used for setting cytometer and experiment group that is the real measuring and studying object.
(Note: The experiment group must incorporate controls of experimental conditions; The control group usually contain unstained and single stained control cells which must go through the same experiment treatments)

4. Precondition cells for subsequent staining if needed. Cell pre-staining processing may include fixation and permeabilization typically.

5. Adjust cell concentration and suspension volume, and add into correct fluorescent staining reagent to obtain expected or optimized fluorescent probe concentration.

6. Avoid light and incubate the staining suspension at optimized or planned temperature and time condition. The best staining conditions always may need to be found out by a series of pretests.
(Note: It may be necessary to mix cells regularly during incubation.)

7. Wash cells and stain them with other fluorescent probes if needed.
(Note: It is very necessary to exclude dead cells by using live/dead dyes for the detecting and/or sorting of rare cell population and intracellular staining cells.)

8. Wash cell by centrifugation 1-3 times, adjust final cell concentration and volume of the washed suspension. Preserve samples in dark at 4℃ or on ice, and run sample in shortest time.
(Note:a. For the results of flow cytometry experiments, the importance of sample quality is greater than 60% at least! b. Typical cell concentration is 1X106 cells/mL for analyzing. For cell sorting, sample concentration can be increased to 5-10 X106 cells/mL, but if cell size is quite big and/or cells tend to aggregate, this number should be reduced accordingly.)

9. If can’t be measured within the same day, samples that labeled with fluorescently conjugated antibodies can be fixed by PFA, washed and stored in dark at 4℃. It may be preserved for about 3 days without obvious changes in fluorescence intensity. Another possible option is to fix the cells but label them at the day they can be run on cytometer. Sorting of living cells must be finished as short a time as possible within the same day!

10. Run samples on a flow cytometer that passed the daily quality control. Correctly optimize the detector voltages or gains to ensure optimal measurement of dim fluorescence, but avoid positive cells exceeding the dynamic range.

11. Measuring single staining control samples to accurately calculate the values of fluorescent compensation, if necessary.
(Note: Step 10 and 11 are performed to optimize parameter settings, and they may be repeatedly adjusted. It should be noticed that it isn’t necessary to change an optimized voltage for pursuing lower compensation values( <±25v may be acceptable).)

12. For the same group of experiment samples, leave all settings unchanged, collect and save data of each sample tubes.
(Note: Remember to acquire sufficient cell number of data. For a consistent statistic accuracy,usually the cell number of each subpopulation in data,even rare cells, should not be less than 1000; as for very limited samples, the cell number of each population in data had better be more than 100.)

13. If do cell sorting, accurately calculate the drop delay time,correctly set sorting conditions and begin sorting.
(Note: a. To turn on the temperature control component of the cytometer and set to 4℃ will be beneficial to keeping a relatively higher viability and reduce changes of gene expression profile. b. Choose sorting nozzle of proper size corresponding to cell size;Select sort mode according to the goal of experiment so as to optimize sorting performance.)

14. The cells collected during sorting should be handled immediately after sorting.
(Note: Cells suffer certain impacts during flow cytometry sorting process. A 4-8 hours’ recovery culture would certainly bring cells to almost the same state just before sorting.)

15. Analyze the data, extract and interpret underlying biological messages according to the experiment design.
(Note: The control samples are essential basis for properly judging and understanding the experiment data.)

* For the result of a flow cytometry experiment, the importance of sample is greater than 60%, especially for cell sorting. Please remember that “Garbage in, Garbage out.”






 
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