Influx cytometer

Manufactured by BD

Sourced in United States

The Influx cytometer is a laboratory instrument used for the analysis and sorting of cells or particles in a sample. It functions by passing the sample through a laser or other light source, which interacts with the cells or particles and produces signals that are detected and analyzed. This information can be used to characterize the properties of the cells or particles, such as size, granularity, and the presence of specific markers.

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Lab products found in correlation

Multicycle, by Phoenix Pharmaceuticals (7 mentions) Lsm 780, by Zeiss (4 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Lsr 2, by BD (2 mentions) Csu spinning disk confocal microscope, by Yokogawa (2 mentions) Iscove s modified dulbecco s medium, by Thermo Fisher Scientific (1 mentions) Ribonuclease, by Merck Group (1 mentions) Facscalibur, by BD (1 mentions) Facsdiva 9, by BD (1 mentions) Countess 2 automated cell counter, by Thermo Fisher Scientific (1 mentions) Chromium chip, by 10x Genomics (1 mentions) Hiseq 2000, by Illumina (1 mentions) Moflo xdp, by BD (1 mentions) Fixable fluorescent viability stain, by Thermo Fisher Scientific (1 mentions) Facsaria 2 cytometer, by BD (1 mentions) Multicycle software program, by Phoenix Pharmaceuticals (1 mentions) Qiamp dna micro kit, by Qiagen (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) M4287, by Merck Group (1 mentions) A1345, by Merck Group (1 mentions)

32 protocols using influx cytometer

Isolation and Sorting of Human Tonsil Lymphocytes

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The Institutional Review Board at Weill Cornell Medical College approved all use of human tissue (IRB#0805009767). All individuals provided informed consent and no compensation n was provided to tissue donors. Primary cells were isolated from fresh deidentified human tonsillectomy specimen tonsillar lymph nodes by density gradient centrifugation and cryopreserved in 90% fetal bovine serum (FBS) with 10% DMSO. Cellular populations were sorted on a BD Influx cytometer. Prior to cell sorting, lymphocytes were resuspended in 90% Iscove’s Modified Dulbecco’s Medium (12330053, Thermo Fisher Scientific) with 10% FBS for 1 hour at 37°C. Lymphocytes were then labeled with anti-CD20 (0.01 dilution), anti-CD10 (0.05 dilution), anti-CD44 (0.025 dilution), anti-CD27 (0.03 dilution), anti-CD38 (0.03 dilution), anti-IgD (0.03 dilution), and anti-CXCR4 (0.1 dilution) conjugated flourochromes (Supplementary Table 1). DAPI was used to exclude non-viable cells. All antibodies used were commercially available and validated by the manufacturers and used according to the manufacturer’s instructions. When possible, positive and negative control populations were used for validation of the antibody.

Doane A.S., Chu C.S., Giammartino D.C., Rivas M.A., Hellmuth J.C., Jiang Y., Yusufova N., Alonso A., Roeder R.G., Apostolou E., Melnick A.M, & Elemento O. (2021). OCT2 pre-positioning facilitates cell fate transition and chromatin architecture changes in humoral immunity. Nature immunology, 22(10), 1327-1340.

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Cell Cycle Analysis by PI and Hoechst Staining

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Propidium iodide (PI) staining was used for cell cycle analysis in fixed cells. Cells were trypsinized and washed twice with PBS before fixation with 70% ice-cold ethanol. After 24 hours, samples were washed with PBS and incubated overnight with ribonuclease (0.3 mg/ml) (Sigma) and PI (15 μg/ml) (Sigma). Samples were analyzed using a BD FACSCalibur with a 488-nm laser and an FL2 detector. For cell cycle analysis of live cells, Hoechst 33342 (Thermo Fisher Scientific) staining was used. A total of 1 × 10⁶ cells were incubated in 1 ml of complete ESC media with Hoechst 33342 (10 μg/ml) at 37°C for 15 min. PI (1 μg/ml) was added to stain dead cells. Cell cycle analysis and sorting were performed with a BD Influx cytometer using an ultraviolet 350-nm laser for the Hoechst staining. All flow cytometry data were analyzed using FlowJo.

Frade J., Nakagawa S., Cortes P., di Vicino U., Romo N., Lluis F, & Cosma M.P. (2019). Controlled ploidy reduction of pluripotent 4n cells generates 2n cells during mouse embryo development. Science Advances, 5(10), eaax4199.

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Lentiviral CRISPRi Screening Protocol

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Individual gRNA oligos were ordered from IDT and cloned into pLKO5.C+E.sgRNA.EFS.GFP for qPCR, ATAC-seq, RNA-seq, HiChIP, and drop-out experiments. gRNA sequences are listed in Supplementary Table 2. gRNA constructs were lentivirally transduced into OCI-Ly7 or SU-DHL-4 CRISPRi cell lines. GFP+ cells were isolated by FACS on a BD Influx cytometer using BD FACSDiva 9.0 software 2 days after transduction. For drop-out curves, cells were infected with GFP-tagged constructs at an infection rate of 20–50% and the fraction of GFP positive cells was monitored by FACS every 3–4 days using FlowJo version 9. The fraction of GFP positive cells was normalized to the fraction of GFP positive cells at day 3–4. Custom R code was used for analysis of FACS data and plotting of results.

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Profiling Senescent Fibroblast Phagocytic Markers

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The cell surface molecules analyzed were MHC-I (PE-Anti H-2 clone M1/42 by Biolegend, San Diego, CA, USA), Calreticulin (Anti-CalR clone PA3-900 by Thermo Fisher, San Diego, CA, USA) and its secondary PerCP-anti-rabbit IgG (ImmunoResearch Laboratories, West Grove, PA, USA), and CD47 (APC-Anti-CD47 clone miap301, Biolegend, San Diego, CA, USA). The cells were washed with PBS 1X, and then 10,000 cells were acquired on the BD Influx cytometer. The cytometry tests were performed on the same day for the NS, SR, and SIPS cells.
The multiplex flow cytometry characteristics used to evaluate the expression of phagocytic function regulatory signals on unfixed and/or permeabilized senescent fibroblasts were the following Fluorophores: FITC, fluorescein isothiocyanate; BV510, Brillant Violet 510; PE-Cy7, phycoerythrin cyanine 7; APC, allophycocyanin; PE, phycoerythrin. The median fluorescence intensity (MdFI) of three surface molecules was compared between NS, RS, and SIPS fibroblast.

Hernández-Mercado E., Prieto-Chávez J.L., Arriaga-Pizano L.A., Hernández-Gutierrez S., Mendlovic F., Königsberg M, & López-Díazguerrero N.E. (2021). Increased CD47 and MHC Class I Inhibitory Signals Expression in Senescent CD1 Primary Mouse Lung Fibroblasts. International Journal of Molecular Sciences, 22(19), 10215.

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Neutral Lipid Staining in Microalgae

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Cells in log (~ 1 × 10⁷ cell mL⁻¹) and stationary (~ 1 × 10⁸ cell mL⁻¹) phases were diluted to a concentration of 1 × 10⁶ cells mL⁻¹ and then stained for neutral lipids using Bodipy 505/515 at 0.12 μg mL⁻¹ and permeabilized with DMSO 20%. After 5 min of incubation in the dark, the samples were acquired in the BD influx cytometer or observed by epifluorescence microscopy.

Osorio H., Jara C., Fuenzalida K., Rey-Jurado E, & Vásquez M. (2019). High-efficiency nuclear transformation of the microalgae Nannochloropsis oceanica using Tn5 Transposome for the generation of altered lipid accumulation phenotypes. Biotechnology for Biofuels, 12, 134.

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Quantifying Cyanophage and Infected Prochlorococcus

Cited 3 times

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Samples for virus abundance and infection were collected every 4 h at 15 m depth using a CTD-rosette equipped with 12 L niskin bottles as described in ref. ^{22 (link)}. Samples for cyanophage abundances (40 mL) were filtered through a 0.2 μm syringe top filter, flash frozen, and stored at −80 ^∘C. Samples for infected cells (40 mL) were filtered through a 20 μm nylon mesh, fixed with electron microscopy grade glutaraldehyde (0.125% final concentration), incubated for 30 minutes in the dark at 4 ^∘C, flash frozen, and stored at −80 ^∘C. Cyanophage concentrations were analyzed using the polony method for T7-like^{78 (link)} or T4-like^{79 (link)} cyanophage families. Virally infected Prochlorococcus was quantified using the iPolony method^{22 (link)} in which Prochlorococcus cells were sorted with a BD Influx cytometer and screened for intracellular T4-like and T7-like cyanophage DNA.

Beckett S.J., Demory D., Coenen A.R., Casey J.R., Dugenne M., Follett C.L., Connell P., Carlson M.C., Hu S.K., Wilson S.T., Muratore D., Rodriguez-Gonzalez R.A., Peng S., Becker K.W., Mende D.R., Armbrust E.V., Caron D.A., Lindell D., White A.E., Ribalet F, & Weitz J.S. (2024). Disentangling top-down drivers of mortality underlying diel population dynamics of Prochlorococcus in the North Pacific Subtropical Gyre. Nature Communications, 15, 2105.

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Electrical Gradient Measurement Using DiBAC4(3)

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The electrical gradient across the membrane was determined using electrical-gradient dependent uptake of bis-(1,3-dibarbituric acid)-trimethine oxonol (DiBAC₄(3))^{8 (link)}. Stationary phase cultures were incubated for 10–20 min with 10 µg ml⁻¹ DiBAC₄(3). Single-cell uptake was measured on a BD influx cytometer (ex. 488 nm, em. 530/40 nm; 100,000 cells) and analyzed using FlowJo v10.3 (FlowJo, LLC). As control, samples were incubated for 1 h with 500 µM of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) prior to staining with DiBAC₄(3).

Van den Bergh B., Schramke H., Michiels J.E., Kimkes T.E., Radzikowski J.L., Schimpf J., Vedelaar S.R., Burschel S., Dewachter L., Lončar N., Schmidt A., Meijer T., Fauvart M., Friedrich T., Michiels J, & Heinemann M. (2022). Mutations in respiratory complex I promote antibiotic persistence through alterations in intracellular acidity and protein synthesis. Nature Communications, 13, 546.

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Single-cell RNA Sequencing for Cell Profiling

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Single-cell RNA sequencing libraries were generated at the JP Sulzberger Columbia Genome Center using a 10X Genomics Chromium Controller and Single-cell 3’ Library & Gel Bead Kit v2 (10X Genomics, #120237). Single cells were sorted in a BD Influx cytometer and were pelleted by centrifugation (300rcf, 5min) followed by resuspension in DMEM at approximately 500cells/μl. Cell viability and concentration was verified using a Countess II Automated Cell Counter (ThermoFisher, #AMQAX1000). Each sample was loaded into one well of a Chromium chip (10X Genomics, #120236), following manufacturer’s instructions, and aiming for a recovery of 5,000 cells per sample. Library construction was carried out according to the manufacturer’s instructions and were sequenced on Illumina Hiseq 2000. The sequenced reads were processed through the Cell Ranger (10x Genomics) pipeline to generate the single-cell gene expression profile.

Arumugam K., Shin W., Schiavone V., Vlahos L., Tu X., Carnevali D., Kesner J., Paull E.O., Romo N., Subramaniam P., Worley J., Tan X., Califano A, & Cosma M.P. (2020). The Master Regulator protein BAZ2B can reprogram human hematopoietic lineage-committed progenitors into a multipotent state.. Cell reports, 33(10), 108474.

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Single-Cell Tumor Sample Fixation and Flow Cytometry

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Once processed, single-cell suspension tumor samples were incubated with a fixable fluorescent viability stain (Life Technologies) for 20 mins (diluted 1:1000 in PBS) prior to incubation with conjugated primary antibodies for 30 mins at 4 °C. Antibodies were diluted in PBS with 0.5% bovine serum albumin (BSA). Stained samples were sorted, using the MoFlo XDP or BD Influx cytometer system.

Mahata B., Pramanik J., van der Weyden L., Polanski K., Kar G., Riedel A., Chen X., Fonseca N.A., Kundu K., Campos L.S., Ryder E., Duddy G., Walczak I., Okkenhaug K., Adams D.J., Shields J.D, & Teichmann S.A. (2020). Tumors induce de novo steroid biosynthesis in T cells to evade immunity. Nature Communications, 11, 3588.

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Cell Harvesting and Flow Cytometry

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Cells were harvested and passed through a 70-µm strainer. The single cells were stained with antibodies against indicated cell surface markers and DAPI, and then analyzed on a BD LSRFortessa cytometer or FACS Aria II cytometer. Cell sorting was performed by the flow cytometry core facilities of the Salk Institute and UCSD on a FACS Aria II cytometer or a BD Influx cytometer. Figures exemplifying the gating strategy are provided in Supplementary Data 7.

Yuan B., Zhou X., Suzuki K., Ramos-Mandujano G., Wang M., Tehseen M., Cortés-Medina L.V., Moresco J.J., Dunn S., Hernandez-Benitez R., Hishida T., Kim N.Y., Andijani M.M., Bi C., Ku M., Takahashi Y., Xu J., Qiu J., Huang L., Benner C., Aizawa E., Qu J., Liu G.H., Li Z., Yi F., Ghosheh Y., Shao C., Shokhirev M., Comoli P., Frassoni F., Yates JR I.I.I., Fu X.D., Esteban C.R., Hamdan S., Izpisua Belmonte J.C, & Li M. (2022). Wiskott-Aldrich syndrome protein forms nuclear condensates and regulates alternative splicing. Nature Communications, 13, 3646.

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