Fluorescence activated cell sorting /flow cytometer facility

Fluorescence activated cell sorting/Flow cytometer facility enables analysis & sorting of heterogeneous mixture of biological cells depending upon the specific light scattering characteristics and/or fluorescent characteristics of each cell in population.

Make and Model

Becton Dickinson (BD), BD FACS Aria SORP (FACS1) & BD FACS Aria Fusion (FACS2)

Available mode for use

    •  Cell/particle analysis and Cell segregation based on scatter/ fluorescence properties

Specifications/Features

  • Designed to sort cells at high flow speed

  • The sorted cells can be collected in a varietyof vessels including 15ml Falcon tubes for 2-waysort, 5ml FACS tubes for 4-way or 2-way sort & 12 well, 24 well, 48 well & 96 well plates for plate sort. The collection vessels should contain media or buffer

    • Lasers:

    FACS1 (4 lasers) -UV laser (355nm) Blue (488nm), Yellow Green (561nm) & Red laser (633nm)

    FACS2 (5 lasers) - UV laser (355nm) Blue (488nm), Yellow Green (561nm), Red laser (637nm) & Violet (405).

    • Detectors:

    FACS1- 13 parameters can be detected including scatter properties.

    FACS2 – 20 parameters can be detected including scatter properties

Application

  • Following are the typical applications of the machine 

    Cell cycle analysis

  • Apoptosis assays

  • Immuno-phenotyping

  • Intra-cellular staining

  • Cell viability assay

Facility in-charge

Contact Email

facs[at] iitb[dot] ac[dot] in

Contact no.

FACS1: 4209/4685 FACS2: 6746

Location

FACS1: Room No 113, CRNTS, IIT Bombay, Powai, Mumbai – 400076

FACS2: Room no. 005, Central Instrumentation room, ground floor, Biosciences &    
              Bioengineering department, IIT Bombay, Powai, Mumbai – 400076

Other contact person(s)

  • FACS1: Ms. Madhura M.Joshi
  • FACS2: Ms.Shikha Gupta

Registration Link:

Submit New Request

Information-IIT Bombay users

Charges for internal users

Information-External users

External user Registration information 8-1-2024.pdf164.25 KB

Registration form-External users

External User Requisition form 8.9.2022 (1).pdf261.74 KB
Technical Specifications

1. FACS1 optical configuration:

Image removed.

Note: UV laser for FACS1 is not working. Hence, 525/50 & 450/50 detector range  (λ) cannot be detected.

2. FACS2 optical configuration:

 

Image removed.

 

FACS1 & FACS2 from Becton Dickinson are sorter for analyzing and sorting fluorescent labelled cells/particles.

FACS1 & FACS2 are specially designed to sort cells at high flow speed.

The sorted cells can be collected in a variety of vessels including 15ml Falcon tubes for 2-waysort, 5ml FACS tubes for 4-way or 2-way sort & 12 well, 24 well, 48 well & 96 well plates for plate sort. The collection vessels should contain media or buffer.

FACS is a sophisticated high throughput instrument. It enables sorting of heterogeneous mixture of biological cells depending upon the specific light scattering characteristics and/or fluorescent characteristics of each cell in population.

A beam of light hits hydrodynamically focused sample stream. Combination of different filters and detectors collect the scatterd light from sample.

1. How does FACS work?

FACS is Fluorescence-Activated Cell sorting. It is a specialized type of flow Cytometry. It provides a method for analyzing & sorting a heterogeneous mixture of cells/particles based on specific light scattering and fluorescence characteristics of each cell/particle. For more details see Shapiro H., 2003 or Adan et al.,2016

2. What kind of samples can be analyzed by FACS?

Biological Cells or particles

3. Is there any restriction on sample size?

The instrument is equipped to normally handle cells/particles in the size range 0.2 to 25 microns. In certain cases, it may be possible for the instrument to handle <0.2 micron particles (NOT cells).

4. How do I book a slot to use the machine?

At https://drona.ircc.iitb.ac.in/ircc/NewFac/CentralFacilities.jsp

5. What do I do with these raw data files?

Raw data files (.fcs) can be analyzed by any flow data analysis software.For details see:Lugli E et al., 2010 & Aghaeepour et al., 2013.

6. Is this system open for External user?

Yes, it is open for external users. For further details visit http://www.ircc.iitb.ac.in/IRCCWebpage/rnd/CentralFacilityRegistration.jsp or contact facs [at] iitb [dot] ac [dot] in

7. I booked a slot, but my samples are not ready. What should I do?

Contact the operator (email facs [at] iitb [dot] ac [dot] in or call 4209/4685) at least 2 hrs before the slot commences

If you have any queries, feel free to drop an email to facs [at] iitb [dot] ac [dot] in

Publications using data from this facility

2010:

Chatterji, B. P., Banerjee, M., Singh, P. & Panda, D. HMBA depolymerizes microtubules, activates mitotic checkpoints and induces mitotic block in MCF-7 cells by binding at the colchicine site in tubulin. Biochem. Pharmacol. 80, 50–61 (2010).

Banerjee, M., Singh, P. & Panda, D. Curcumin suppresses the dynamic instability of microtubules, activates the mitotic checkpoint and induces apoptosis in MCF-7 cells. FEBS J. 277, 3437–3448 (2010).

Hema Sagar, G., Tiwari, M. D. & Bellare, J. R. Flow cytometry as a novel tool to evaluate and separate vesicles using characteristic scatter signatures. J. Phys. Chem. B 114, 10010–10016 (2010).

2011:

Tiwari, M. D., Mehra, S., Jadhav, S. & Bellare, J. R. All-trans retinoic acid loaded block copolymer nanoparticles efficiently induce cellular differentiation in HL-60 cells. Eur. J. Pharm. Sci. 44, 643–652 (2011).

 Kini, S., Bahadur, D. & Panda, D. Magnetic PLGA nanospheres: A dual therapy for cancer. IEEE Trans. Magn. 47, 2882–2886 (2011).

2012:

Tiwari, M. D., Sagar, G. H. & Bellare, J. R. Flow cytometry-based evaluation and enrichment of multiwalled carbon nanotube dispersions. Langmuir 28, 4939–4947 (2012).

2013:

Gajula, P. K., Asthana, J., Panda, D. & Chakraborty, T. K. A synthetic dolastatin 10 analogue suppresses microtubule dynamics, inhibits cell proliferation, and induces apoptotic cell death. J. Med. Chem. 56, 2235–2245 (2013).

Rai, A. et al. CXI-benzo-84 reversibly binds to tubulin at colchicine site and induces apoptosis in cancer cells. Biochem. Pharmacol. 86, 378–391 (2013).

2014:

Singh, D. et al. SB-RA-2001 inhibits bacterial proliferation by targeting FtsZ assembly. Biochemistry 53, 2979–2992 (2014).

Kar, R. K. et al. Stochastic galactokinase expression underlies GAL gene induction in a GAL3 mutant of Saccharomyces cerevisiae. FEBS J. 281, 1798–1817 (2014).

Mummidivarapu, V. V. S., Yarramala, D. S., Kondaveeti, K. K. & Rao, C. P. Time- and concentration-dependent reactivity of Cys, Hcy, and GSH on the diels-alder-grafted 1,3,5-tris conjugate of calix[6]arene to bring selectivity for Cys: Spectroscopy, microscopy, and its reactivity in cells. J. Org. Chem. 79, 10477–10486 (2014).

2015:

Reddy, P. J. et al. A comprehensive proteomic analysis of totarol induced alterations in Bacillus subtilis by multipronged quantitative proteomics. J. Proteomics 114, 247–262 (2015).

Kini, S., Bahadur, D. & Panda, D. Mechanism of anti-cancer activity of benomyl loaded nanoparticles in multidrug resistant cancer cells. J. Biomed. Nanotechnol. 11, 877–889 (2015).

Carvalho, E., Verma, P., Hourigan, K. & Banerjee, R. Myocardial infarction: Stem cell transplantation for cardiac regeneration. Regen. Med. 10, 1025–1043 (2015).

Gupta, J., Bhargava, P. & Bahadur, D. Fluorescent ZnO for imaging and induction of DNA fragmentation and ROS-mediated apoptosis in cancer cells. J. Mater. Chem. B 3, 1968–1978 (2015).

Prajapat, M. K., Jain, K. & Saini, S. Control of MarRAB operon in Escherichia coli via autoactivation and autorepression. Biophys. J. 109, 1497–1508 (2015).

Jaiswal, M. K. et al. Magneto-thermally responsive hydrogels for bladder cancer treatment: Therapeutic efficacy and in vivo biodistribution. Colloids Surfaces B Biointerfaces 136, 625–633 (2015).

Sanganeria, P., Chandra, S., Bahadur, D. & Khanna, A. Effect of HSA coated iron oxide labeling on human umbilical cord derived mesenchymal stem cells. Nanotechnology 26, 125103 (2015).

2016:

Kalantre, G. S. & Viswanathan, G. A. Reconstruction of ensemble of single-cell time trajectories from discrete-time fluorescence data: Oscillatory MAPK dynamics. IFAC-PapersOnLine 49, 184–189 (2016).

Gupta, J., Mohapatra, J., Bhargava, P. & Bahadur, D. A pH-responsive folate conjugated magnetic nanoparticle for targeted chemo-thermal therapy and MRI diagnosis. Dalt. Trans. 45, 2454–2461 (2016).

Jain, K. & Saini, S. MarRA, SoxSR, and Rob encode a signal dependent regulatory network in Escherichia coli. Mol. Biosyst. 12, 1901–1912 (2016).

Poojari, R., Kini, S., Srivastava, R. & Panda, D. Intracellular interactions of electrostatically mediated layer-by-layer assembled polyelectrolytes based sorafenib nanoparticles in oral cancer cells. Colloids Surfaces B Biointerfaces 143, 131–138 (2016).

Sasidharan, S., Bahadur, D. & Srivastava, R. Protein-Poly(amino acid) Nanocore-Shell Mediated Synthesis of Branched Gold Nanostructures for Computed Tomographic Imaging and Photothermal Therapy of Cancer. ACS Appl. Mater. Interfaces 8, 15889–15903 (2016).

Sasidharan, S., Bahadur, D. & Srivastava, R. Albumin stabilized gold nanostars: A biocompatible nanoplatform for SERS, CT imaging and photothermal therapy of cancer. RSC Adv. 6, 84025–84034 (2016).

2017:

Gupta, J., Mohapatra, J. & Bahadur, D. Visible light driven mesoporous Ag-embedded ZnO nanocomposites: reactive oxygen species enhanced photocatalysis, bacterial inhibition and photodynamic therapy. Dalt. Trans. 46, 685–696 (2017).

2018:

Mohite, G. M. et al. Comparison of Kinetics, Toxicity, Oligomer Formation, and Membrane Binding Capacity of α-Synuclein Familial Mutations at the A53 Site, Including the Newly Discovered A53V Mutation. Biochemistry 57, 5183–5187 (2018).

Kumar, R. et al. Cytotoxic Oligomers and Fibrils Trapped in a Gel-like State of α-Synuclein Assemblies. Angew. Chemie - Int. Ed. 57, 5262–5266 (2018).

Surve, M. V. et al. Heterogeneity in pneumolysin expression governs the fate of Streptococcus pneumoniae during blood-brain barrier trafficking. PLoS Pathog. 14, 1–29 (2018).

Nag, R., Kandi, R. & Rao, C. P. Host-Guest Complexation of a Lipoic Acid Conjugate of Calix[4]arene with Pyridinium Moiety on Gold Nanorods for Mitochondrial Tracking Followed by Cytotoxicity in HeLa Cells under 633 nm Laser Light. ACS Sustain. Chem. Eng. 6, 8882–8890 (2018).

Chandan, R. & Banerjee, R. Pro-apoptotic liposomes-nanobubble conjugate synergistic with paclitaxel: A platform for ultrasound responsive image-guided drug delivery. Sci. Rep. 8, 1–15 (2018).

Laxmi, V., Tripathi, S., Joshi, S. S. & Agrawal, A. Microfluidic Techniques for Platelet Separation and Enrichment. J. Indian Inst. Sci. 98, 185–200 (2018).

Upadhyay, A., Kandi, R. & Rao, C. P. Injectable, Self-Healing, and Stress Sustainable Hydrogel of BSA as a Functional Biocompatible Material for Controlled Drug Delivery in Cancer Cells. ACS Sustain. Chem. Eng. 6, 3321–3330 (2018).

2019:

Parundekar, A., Kalantre, G., Khadpekar, A. & Viswanathan, G. A. Operating regimes in a single enzymatic cascade at ensemble-level. PLoS One 14, (2019).

Nirmala, J. G., Beck, A., Mehta, S. & Lopus, M. Perturbation of tubulin structure by stellate gold nanoparticles retards MDA-MB-231 breast cancer cell viability. J. Biol. Inorg. Chem. 24, 999–1007 (2019).

Nirmala, J. G. & Lopus, M. Tryptone-stabilized gold nanoparticles induce unipolar clustering of supernumerary centrosomes and G1 arrest in triple-negative breast cancer cells. Sci. Rep. 9, (2019).

Manohar, S. et al. Combining fluorescent cell barcoding and flow cytometry-based phospho-ERK1/2 detection at short time scales in adherent cells. Cytom. Part A 95, 192–200 (2019).

2020:

Prasad, C., Bhatia, E. & Banerjee, R. Curcumin Encapsulated Lecithin Nanoemulsions: An Oral Platform for Ultrasound Mediated Spatiotemporal Delivery of Curcumin to the Tumor. Sci. Rep. 10, 1–16 (2020).

Bhatia, E. & Banerjee, R. Hybrid silver-gold nanoparticles suppress drug resistant polymicrobial biofilm formation and intracellular infection. J. Mater. Chem. B 8, 4890–4898 (2020).

Prabhuraj, R. S., Bomb, K., Srivastava, R. & Bandyopadhyaya, R. Selection of superior targeting ligands using PEGylated PLGA nanoparticles for delivery of curcumin in the treatment of triple-negative breast cancer cells. J. Drug Deliv. Sci. Technol. 57, 101722 (2020).

Patel, Y., Rai, D., Das, K., Dhandayuthapani, S. & Mehra, S. Ethanol in Combination with Oxidative Stress Significantly Impacts Mycobacterial Physiology. J. Bactriology 202, (2020).

Laxmi, V., Tripathi, S., Joshi, S. S. & Agrawal, A. Separation and Enrichment of Platelets from Whole Blood Using a PDMS-Based Passive Microdevice. Ind. Eng. Chem. Res. 59, 4792–4801 (2020).

Sharma, S., Chatterjee, A., Kumar, P., Lal, S. & Kondabagil, K. Upregulation of miR-101 during influenza A virus infection abrogates viral life cycle by targeting mTOR pathway. Viruses 12, (2020).

2021:

Chandna, P. & Gundabala, V. Pillar based microfluidic approach to sorting of microparticle mixtures at various particle ratios. Microfluid. Nanofluidics 25, (2021).

Kar, N., Gupta, D. & Bellare, J. Ethanol affects fibroblast behavior differentially at low and high doses: A comprehensive, dose-response evaluation. Toxicol. Reports 8, 1054–1066 (2021).

Nirmala, J. G., Rachineni, K., Choudhary, S., Hosur, R. V. & Lopus, M. Triphala polyphenols-functionalized gold nanoparticles impair cancer cell survival through induction of tubulin dysfunction. J. Drug Deliv. Sci. Technol. 61, (2021).

Gupta, D. & Bellare, J. Highly controlled robotic customized gel functionalization on 3D printed PCL framework for bone tissue engineering. Bioprinting24, (2021).

 

2022:

Biswas, A. et al. Stabilization and fluorescence light-up of G-quadruplex nucleic acids using indolyl-quinolinium based probes. Phys. Chem. Chem. Phys. (2022) doi:10.1039/d1cp04718c

2023:

Barpanda A, Biswas D, Verma A, Parihari S, Singh A, Kapoor S, Kantharia C, Srivastava S. Integrative Proteomic and Pharmacological Analysis of Colon Cancer Reveals the Classical Lipogenic Pathway with Prognostic and Therapeutic Opportunities. J Proteome Res. 2023 Mar 3;22(3):871-884. doi: 10.1021/acs.jproteome.2c00646. Epub 2023 Feb 2. PMID: 36731020.

Conference presentations
18th TNF superfamily meet, 10th-14th October,2021, Switzerland on Unraveling the Intracellular cross-talk governed TNF alpha mediated survival and apoptosis signaling (Presenter:Ms. SharmilaBiswas)

▪ https://pubs.acs.org/doi/10.1021/acs.oprd.2c00356
▪ https://pubs.rsc.org/en/content/articlelanding/2023/re/d2re00405d
▪ https://pubs.rsc.org/en/content/articlelanding/2022/mh/d2mh00244b
▪ https://www.sciencedirect.com/science/article/abs/pii/S0014305722004402

▪ Surabhi Mehra, Sahil Ahlawat, Harish Kumar, Debalina Datta, Ambuja Navalkar, Nitu Singh, Komal Patel, Laxmikant Gadhe, Pradeep Kadu, Rakesh Kumar, Narendra N Jha, Arunima Sakunthala, Ajay S Sawner, Ranjith Padinhateeri, Jayant B Udgaonkar, Vipin Agarwal, Samir K Maji, (2022), α-Synuclein aggregation intermediates form fibril polymorphs with distinct prion-like properties, Journal of Molecular Biology 434(19), 167761

▪ Arunima Sakunthala, Debalina Datta, Ambuja Navalkar, Laxmikant Gadhe, Pradeep Kadu, Komal Patel, Surabhi Mehra, Rakesh Kumar, Debdeep Chatterjee, Jyoti Devi, Kundan Sengupta, Ranjith Padinhateeri, Samir K Maji, (2022), Direct Demonstration of Seed Size-Dependent α-Synuclein Amyloid Amplification, Journal of Physical Chemistry Letters 13(28), 6427–6438

▪ Ambuja Navalkar, Ajoy Paul, Arunima Sakunthala, Satyaprakash Pandey, Amit Kumar Dey, Sandhini Saha, Sarthak Sahoo, Mohit K Jolly, Tushar K Maiti, Samir K Maji, (2022), Oncogenic gain of function due to p53 amyloids occurs through
aberrant alteration of cell cycle and proliferation, Journal of Cell Science 135(15), jcs259500

▪ Mukherjee S, Sawant AV, Prassanawar SS, Panda D. Copper-Plumbagin Complex Produces Potent Anticancer Effects by Depolymerizing Microtubules and Inducing Reactive Oxygen Species and DNA Damage. ACS Omega. 2023 Jan 10;8(3):3221-3235. doi: 10.1021/acsomega.2c06691.

▪ Saha R, Patkar S, Pillai MM, Tayalia P. 2023. Bilayered Skin Substitute Incorporating Rutin Nanoparticles for Antioxidant, Anti-inflammatory and Anti-fibrotic Effect. Biomater. Adv.150:213432.

▪ Hemavathi Dhandapani, Armaan Siddiqui, Shivam Karadkar, Prakriti Tayalia. In vitro 3D spheroid model preserves tumor microenvironment of hot and cold breast cancer subtypes. Advanced Healthcare Materials. (accepted for publication on
29th April 2023)

▪ Sagitha P, Dhandapani H, Tayalia P. Choline ester based ionic liquid: A multifunctional system to enhance nucleic acid stability, drug solubilization and cell penetration. Int J Biol Macromol. 2023 Mar 17;238:124059. doi:
10.1016/j.ijbiomac.2023.124059. Epub ahead of print. PMID: 36934812.

▪ Manohar S*, Shah P*, Biswas S*, Mukadam A, Joshi M, Viswanathan G. Combining fluorescent cell barcoding and flow cytometry‐based phospho‐ERK1/2 detection at short time scales in adherent cells. Cytometry Part A 2019; 95(2):192-200. *Equal contribution Impact Factor: 3.433

▪ Biswas S*, Tikader B*, Kar S and Viswanathan, G Modulation of signaling cross-talk between pJNK and pAKT generates optimal apoptotic response. PLOS Computational Biology 2022; Oct 14;18(10):e1010626. Impact Factor : 4.53
b) 

Minutes of Facility Management Committee

Minutes of the FACS Central Facility FMC Meeting

Date: 12/7/2017

Time: 11:20 AM

Venue: Dept. Library, Chemical Engg.

Present: Anindya Datta, P Jayadeva Bhat, Ganesh Viswanathan (Prof. Jayesh Bellare and Prof. D. Bahadur expressed their inability to attend the meeting due to prior commitments.)

Agenda:

 Overall status: (Matters of Information)

• Sharing of Operator between FACS, Central facility and Biosciences FACS facility

• Samples will be separately analyzed on the respective instruments.

• External samples will be analyzed on FACS, Central facility and the money generated from it will be sent to Central facility account.

• External samples will be analyzed on Biosciences machine only in case, if there is any technical limitation in the FACS Central facility.

 Policy for Handling of Primary samples

• Bioethics Committee approval

• Precautions & Safety requirements

• Hepatitis B vaccination for Staff

• Lab coats, Hand gloves, Ethanol, Sodium Hypochlorite, Biohazard bags to be ordered

• TAs will not handle Primary samples

 Budget FY 2017-2018

Minutes:

1) Committee was apprised of Sharing the duties of the FACS Central facility operator with the BSBE FACS facility

2) Use of secondary cells with active pathogenic substance or primary samples: It was suggested the FACS Central Facility seeks Bioethics committee approval for this purpose. The committee suggested a roster of the kind of samples that can be handled in the facility is created. In addition, the committee also suggested creating a list of different kind of samples that cannot be handled by the facility but can be analyzed in the BSBE FACS facility and that users bringing such samples should be directed to BSBE FACS Facility. Appropriate information regarding such samples should be collected on the DRONA portal and accordingly samples have to allotted to the appropriate facility. (See attached flowchart & decision chart elucidating the allotment decision process.)

It was noted that external samples will continue to be run in FACS Central Facility as the facility, in its current form, is found adequate for the nature of the such samples received so far. However, in future, if certain external samples require access to BSBE FACS Facility due to inadequacy of FACS Central Facility, it will be taken up on a case-to-case basis.

3) Budget: Committee approved the proposed budget of Rs.15,13,450( Consumables - Rs. 3,83,450, AMC charges - Rs.6,50,000 & Man power – 4,80,000)

4) For smooth operation, it was felt that a common sample submission portal for both FACS Central Facility and BSBE FACS facility is necessary. Inclusion of the BSBE FACS Facility in the DRONA sample submission portal for the FACS Central Facility is recommended. Since BSBE FACS Facility is purchased from a grant to the BSBE Department, users from BSBE department will have a different tariff. (See flowchart for incorporation of this as well on DRONA.)

Signature of Facility Management Committee members

 

 

 

Prof. Ganesh Viswanathan                                                                Prof. Anindya Datta

(Convener – FACS Central facility)                                                     (Head – CRNTS)

 

 

 

Prof. D. Bahadur                                                                                    Prof. P J Bhat                                                                                  Prof. Jayesh

Instructions for sample preparation/submission
  • Type/Toxicity/Pathogenecity of the samples and precautions during sample analysis and sample disposal need should be discussed with the operator/TAs before booking the slot.
  • Samples should not be viscous.
  • Samples should not be prepared in Organic solvents (e.g. THF, Acetone, Hexane, Toluene etc)
  • Sample should be a single cell suspension.Clumping should be avoided. Clumps can block nozzle and can affect analysis.
  • Minimum volume of sample required for acquisition is 500ul and Sample density should be approx. 1x106 cells or particles per ml for better analysis.
  • User should bring appropriate compensation controls required for the experiment.
  • The users are expected to bring their own samples for analysis.
  • The user should bring an unstained sample for each experiment.
Instructions for Users
  • First time users should contact the operator to understand the requirements.
  • Most of the slots in a typical day are booked. Moreover, samples to be analyzed are time bound. Please adhere to the slots allotted. In case, there may be a delay in bringing the samples, the operator must be informed at least 3 hours before the slot commences. Not turning up without informing the operator would lead to ban of usage of machine for 1 month.
  • The user should bring a new blank CD every time to collect the flow cytometry data. USB drives are strictly not allowed due to virus threat.
  • For new samples or experiments, user is expected to bring one or two literature reference papers if available.
  • Flow Cytometry data used for publication should appropriately acknowledge  IRCC and FACS Central facility.
Instructions for Registration
  • The requisition form is available online.
  • Users should submit duly filled registration forms online only. Hard copies will not be accepted.
  • System generated mail will be sent to user's Id about the date & time of the slot that has been allotted.

Usage Charges