Flow Cytometry Facility

Forthcoming: [Workshop]: “Applications of laser flow cytometry in Biomedical Research 

Visit http://fcm2016.in/ for more details. 


The Flow Cytometry Facility, located in the C-Wing, Ground Floor of the Division of Biological Sciences is a dedicated scientific service offering an extensive flow cytometry analysis and sorting facility with state of the art instrumentation, operated by qualified personnel including project assistants, research scientists and scientific officers. It is actively involved in acquisition, analyses and cell sorting in various areas of bio-medical research catering to all the departments of the Indian Institute of Science. It is extensively used by the research community from not only the departments of Division of Biological Sciences, but also from departments like Materials Research Centre (MRC), Materials Engineering (ME), Mechanical Engineering, Centre for Neuroscience (CNS), Inorganic Physical Chemistry (IPC), CIDR, Organic Chemistry.

That researchers increasingly recognize the contributions of this facility is reflected by a striking increase in the number of activities and projects that it has been supporting since 2014 onwards.

THE ABILITY TO DISCRIMINATE, QUANTIFY AND SORT, DISTINCT POPULATIONS OF CELLS /ORGANELLES/ PARTICLES BASED ON THEIR EXPRESSION OF SPECIFIC MARKERS WITH RAPID PACE IS FLOW CYTOMETRY.

SCIENTIFIC STAFF

Convenor: Prof. Dipankar Nandi (Biochemistry)

Scientific Officer: Dr. William Surin (Microbiology and Cell Biology)

Research Scientist: Dr. Uttara Chakraborty (September, 2014 till date)

Project Assistants:

  • Vasista Adigas (Oct 2013- July 2015)
  • Dharani Manoj (Aug 2014 - July 2015)
  • Urvashi Chauhan (Mar 2014- till date)
  • Leepika Baid (Oct 2014- till date)
  • Ranjitha R. (July 2015- till date)
  • Ankitha Ramachandra (July 2015- till date)

Scientific PERSONNEL

Prof. Dipankar Nandi, Convenor (Professor, Biochemistry)

Dr. William R. Surin (Senior Scientific Officer, MCB)

Dr. Uttara Chakraborty (Senior Research Scientist, Bio-imaging and Flow Cytometry

Urvashi Chauhan (Project Assistant)

Leepika Baid (Project Assistant)

Ranjitha R. (Project Assistant)

Ankita Ramachandran. (Project Assistant)

The project assistants and research scientist are supported by the IISC-DBT partnership program and partly by the service agreement with the vendor companies of the high-end instruments purchased by the facility.

Equipments:

The facility has 4 analytical cytometers including one plate-reading cytometer and 2 high speed cell sorters. All the instruments are operator-assisted here where the instrument manufacturing companies provide extensive training to the project assistants before they work on them. Users of the instruments are also trained in regular intervals on the technology through workshops and demonstrations held jointly with the major companies into flow cytometry.

Analytical Cytometers:

  • BD FACSVerse: Three lasers (405 nm, 488 nm and 633 nm) and 8 fluorescence detectors. Windows XP OS with FACS Suite software for acquisition and analyses. It also has the plate-reading module as an add-on.

  • BD FACSCanto II: Three lasers (405 nm, 488 nm and 633 nm) and 8 fluorescence detectors. Windows XP OS with FACS Diva ver. 6.0 software for acquisition and analyses.

  • BD FACSCalibur: Two lasers (488 nm and 635 nm) and 4 fluorescence detectors. Mac OS with Cell QuestPro software for acquisition and analyses.

  • BD FACScan: Single laser (488 nm) and 3 fluorescence detectors. Mac OS with Cell QuestPro software for acquisition and analyses.

Cell Sorters: The 2 sorters can retrieve up to 4 specifically defined populations so that cells may be recovered for re-culture, functional assays or RNA or DNA recovery. Sorting experiments are scheduled and planned well in advance with our users before performing on the instruments.

  • BD FACSAria II: Three lasers (375 nm, 488 nm and 633 nm) and 9 fluorescence

detectors. Windows XP OS with FACS Diva ver. 6.0 software for acquisition, analyses and cell sorting.

  • BC MoFlo XDP: Three lasers (350 nm, 488 nm and 640 nm) and 10 fluorescence

detectors. Windows XP OS with Summit software for acquisition, analyses and cell sorting.

Details of all the instruments are available at the facility webpage http://mcbl.iisc.ernet.in/FACS/FACSFacility.html

Flow Cytometry usage guidelines

In order to fulfill the expectations of our users and to ensure smooth functioning of the Divisional Flow Cytometry Facility, we recommend the following guidelines to be considered by all.

1. There are 4 regular slots given per day for every analyzer in the facility. 10-11 am, 11.30-12.30 am, 2-3 pm and 3.30-4.30 pm. Bookings are to be made from the facility via online calendars assigned for individual cytometers.

2. For special time point experiments, the student should come on the day of booking and take the extra slots after sending a formal request mail for continuous slots for more than a week to uttara@biochem.iisc.ernet.in

3. For even further requirements, extra half an hour bookings can be made upon requests with the operator for a maximum of 10 samples, and in this manner 4 extra bookings can be given per lab per month.

4. Per day there can be 1 extra slot provided for each analyzer (BD FACSCanto and BD FACSVerse) in keeping with the usage requirement of the regular slots of the day.

5. In normal course, we can provide maximum slots up to 12 per lab in a month. Further requests for slots should come from the concerned PI.

6. Booking cancellations can be allowed for a maximum of 3 slots per lab. Upon further cancellations the concerned PI will be informed.

7. Students are requested to come with samples on time and report in advance for any kind of delay, at least 15 min before the slot time.

8. Both the analyzers (BD FACSCanto and BD FACSVerse) are equipped with 3 lasers, 405nm, 488nm, and 633nm. Kindly refrain from using only the FACSVerse for all analyses, as performance of both the cytometers are calibrated to the best. The facility can shift few experiments to either analyzer if there is an overload on any one.

9. Sorting experiments must be discussed, planned and scheduled with the core facility employee in advance.

10. All data files are to be taken by the students immediately after the experiments are done. We will remove all files from the systems every month to keep free disk space for proper functioning of the softwares. Data files will be always given in .fcs 3.0 version from all the cytometers and once given to students; the facility will not take responsibility to preserve them for future.

11. Any formal presentations or publications resulting from the work performed in the facility should be duly acknowledged and a reprint/soft copy should be provided to the facility.

12. Users are requested to discuss their experiments with the facility and depending upon their experimental requirements, the cytometers will be assigned to them by the facility. 6-8 color experiments will get priority in BD FACSVerse over 1-2 color experiments, which may be performed on either of the two cytometers, BD FACSCaliber and BD FACSCanto.

The aim of our facility has always been to provide our researchers complete access to the latest flow cytometers and we are hopeful to meet your demands by following the above user guidelines.

Ongoing research areas: 

The goal of the facility is to proactively introduce flow cytometric methods into new research areas while supporting and extending current research. Flow cytometry in this facility is most commonly used for cell cycle analysis, immunophenotyping, cell proliferation or cell death assays, tracking of gene transfer and cell transfection studies and cytokine detection by intracellular staining or by cytometric bead array. We offer a wide range of flow cytometric techniques. Our equipments add flexibility in the execution of experiments allowing different approaches to addressing scientific problems.

Major projects initiated and accomplishments

  • Stable cell line generation from lenti-virus transduced cell lines, requiring several rounds of enrichment/ cell sorting of target populations into 96-well plates. We have used this technique to generate homogeneous cell lines with stably genome-integrated transduced genes and the selection of clones carrying the improved fluorescent protein variants out of genetically engineered libraries.

  • Rare population detection and isolation cancer stem-like cells and flow cytometric detection and sorting of CTCs in breast cancer cell lines.

  • Flow cytometric isolation of human antibodies from a non-immune Saccharomyces cerevisiae surface display library. Sorting of mutagenic scFv library for isolation of higher affinity clones.

  • Flow cytometric analyses of human testicular cells for identification of infertile human patients.

  • Enumeration of innate and adaptive immune cells from human whole blood samples using BD Trucount absolute counting tubes, wherein absolute counts are taken by flow cytometric analyses for different populations such as NK cells, dendritic cells, monocytes, CD4, CD8, B cells and T regulatory cells. This study analyses the cell populations before and after BCG re-vaccination in young adults and is part of a wider clinical study conducted at the Centre for Infectious Disease Research (CIDR). This is one of the major multi-color immunophenotyping assays in the facility where 8 fluorochromes (FITC, PE, PerCP, PE-Cy7, APC, APC-Cy7, V450 and V500) are analyzed per sample.

  • Detection of HPF fluorescence as an indicator of hydroxyl radical during antibiotic treatment of M. smegmatis (mc2 155) and M. tuberculosis (H37Ra, avirulent) strains. Real time measurement of unstable GFP fluorescence as an indicator of bacterial metabolic activity and detection of oxidative stress in bacteria using redox sensitive GFP by measuring ratiometric excitation at two different wavelengths. Subsequently, cell sorting of the two species was performed based on their variable metabolic activity using unstable GFP as a marker. A similar study has assayed antibiotic entry into hypoxic mycobacterial cells by measuring fluorescence within the cells in a time dependent manner.

 

Other services from the facility

The members of the facility are available to provide advice on the design of experiments, sourcing and supply of reagents, data analysis, presentation and interpretation as well as troubleshooting with respect to instrumentation and experiments.

  1. Teaching and outreach:
  • UG students’ visit to our facility as part of IISC Bachelor of Science (Research) program, 4 day teaching and demonstration of the flow cytometers to the UG students- every year in the month of October.
  • Participants of King’s College London credited Summer School on Advanced Concepts in Infection and Immunity- Impact on translational Medicine at St. John’s Research Institute, conducted by Prof. Annapurna Vyakarnam;  visited 23 July, 2015.  
  • Lectures conducted from Oct, 2014-August, 2015
    • Talk by Dr. Darren Ellemor from BD Biosciences on “ Multicolor flow cytometry and cell sorting”- 8th December, 2014
    • Talk by Dr. Hemant Agrawal, Director, FlowCytometry Solutions on Data Analyses in Flow Cytometry-10th Mar, 2015
    • Talk by Dr. Matthias Schiemann, Technische Universität München - Institute for Medical Microbiology, Immunology and Hygiene, Dirk H. Busch,  on “Forensic Flow Cytometry – a functional verification of flow cytometry cell sorting and analysis”- 6th May, 2015
  • Workshops:
  1. BD Biosciences along with the Indian Institute of Science jointly organized a workshop on flow cytometry at the Facility, Division of Biological Sciences from 16-18th March, 2015. The workshop had a series of talks followed by hands-on training to work on the instruments. Important applications such as multicolor flow cytometry, cell sorting, cytokine analyses and/or apoptosis studies were covered in this "Training, Tech. Support and Education (TTE)" Program.
  2. Beckman Coulter along with the Indian Institute of Science jointly organized a workshop on Cell Sorting at the Facility, Division of Biological Sciences from 6-8th July, 2015. As part of the workshop there was a series of talks followed by hands-on training to work on the MoFlo XDP. This workshop involved modules such as rare cell population and side population analyses and bacterial cell sorting. 

 

 2.     Facility Journal Club started from July 2014 as a platform to update the facility personnel with the advances of confocal imaging and flow cytometry. Held jointly with members of the flow cytometry and Bio-imaging team.

ACKNOWLEDGEMENTS

The Flow Cytometry facility being a non-chargeable facility fully acknowledges the DBT Grant-in-aid partnership programme for all financial support. We are also thankful to BD Biosciences for their regular support in terms of training our personnel, servicing and troubleshooting in this field.