Kc57 rd1

Manufactured by Beckman Coulter

Sourced in Germany

The KC57-RD1 is a laboratory instrument designed for automated cell counting and analysis. It utilizes flow cytometry technology to precisely measure and characterize various cell populations within a sample. The core function of the KC57-RD1 is to provide accurate and reliable data on cell count, size, and other relevant parameters.

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Lab products found in correlation

Fixation and permeabilization kit, by Thermo Fisher Scientific (2 mentions) Facscanto 2, by BD (2 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions) Cytofix cytoperm kit, by BD (1 mentions) X tremegene hp, by Roche (1 mentions) Facsuite software, by BD (1 mentions) Facsverse flow cytometer, by BD (1 mentions) Facsverse, by BD (1 mentions) Pe conjugated anti cd80, by BD (1 mentions) Pe conjugated donkey anti rabbit, by Jackson ImmunoResearch (1 mentions) Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (1 mentions) Fitc conjugated goat anti rhesus igg, by Southern Biotech (1 mentions) Viromer red, by BioNTech (1 mentions) D 001810 10 20, by Horizon Discovery (1 mentions) Opti mem, by Thermo Fisher Scientific (1 mentions) Vpd450, by BD (1 mentions) T cell activation expansion kit, by Miltenyi Biotec (1 mentions) Formaldehyde, by Tousimis (1 mentions) Lrsii flow cytometer, by BD (1 mentions) Fixation permeabilization kit, by Thermo Fisher Scientific (1 mentions)

13 protocols using kc57 rd1

Transfection and Immunofluorescence of ACH-2 and CEM Cells

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ACH-2 (Folks et al., 1989 (link)) or CEM cells were cultured with 10 nM Phorbol 12-myristate 13-acetate (PMA) in complete RPMI 1640 medium for three days prior to transfection (Folks et al., 1989 (link); Fujinaga et al., 1995 ). 3 × 10⁶ cells were seeded in 6 well plates. Cells were kept in the same medium after transfection for two days. A total of 2 μg DNA was transfected with X-tremeGene HP (Roche, Basel, Switzerland) into each well. TagBFP transfection mixtures consisted of 0.25 μg plasmids of either CAG-driven αCA-Nb-TagBFP, αCA-dNb^6mut-TagBFP, or dGBP1-TagBFP, along with 1.75 μg pCAG-DsRed. TagRFP transfection mixtures consisted of 0.25 μg plasmids of either CAG-driven αCA-dNb^6mut-TagRFP or C-CA along with 1.75 μg of pCAG-GFP. Cells were fixed in 4% PFA for 30 min at room temperature. Cells were washed twice with 2% heat-inactivated fetal calf serum in PBS, and finally re-suspended in PBS to be used for flow cytometry. For immunofluorescence with flow cytometry, cells were fixed and permeabilized using the CytoFix/CytoPerm kit (BD Biosciences, San Jose, CA). The antibody KC57-RD1 (6604667, Beckman Coulter), which recognizes the 24 kDa protein, also known as CA, of HIV-1 core antigen, was used to detect CA.

Tang J.C., Drokhlyansky E., Etemad B., Rudolph S., Guo B., Wang S., Ellis E.G., Li J.Z, & Cepko C.L. (2016). Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies. eLife, 5, e15312.

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Intracellular HIV-1 p24 Quantification

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For intracellular p24 staining, U1-shCTH and U1-shNT cells were stimulated with PMA (5 ng/ml), washed with PBS followed by fixation and permeabilization using a fixation and permeabilization kit (eBiosciences). Permeabilized cells were then incubated with 50 μl of 1:100 dilution of phycoerythrin (PE)-conjugated mouse anti-p24 monoclonal antibody (KC57-RD1; Beckman Coulter, Inc) for 30 min at room temperature. After incubation, the cells were washed two times and the fluorescence of stained samples was acquired using BD FACSVerse flow cytometer (BD Biosciences). The data were analyzed using FACSuite software (BD Biosciences).

Pal V.K., Agrawal R., Rakshit S., Shekar P., Murthy D.T., Vyakarnam A, & Singh A. (2021). Hydrogen sulfide blocks HIV rebound by maintaining mitochondrial bioenergetics and redox homeostasis. eLife, 10, e68487.

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Quantifying HIV-1 Infection in TZM-bl Cells

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2 × 10⁵ and 4 × 10⁵ TZM-bl cells per well were seeded in 6-well plates and pre-incubated at 37°C with complete DMEM containing DMSO or 6 µM APC for 24 hr. Cells were infected with HIV-1 or HIV-1 (IN.eGFP), respectively, with an m.o.i. of 0.1 in the presence of DMSO or 6 µM APC. At 4 hr p.i., medium was replaced by DMEM and incubation was continued. At 48 hr p.i., cells were fixed with 3% PFA, permeabilized and probed with phycoerythrin-conjugated monoclonal anti-HIV CA antibody KC57-RD1 (Beckman Coulter, Germany). The proportion of infected cells was quantified by flow cytometry using a BD FACSVerse instrument.

Peng K., Muranyi W., Glass B., Laketa V., Yant S.R., Tsai L., Cihlar T., Müller B, & Kräusslich H.G. (2014). Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid. eLife, 3, e04114.

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Profiling Dendritic Cell Activation and HIV-1 Infection

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DCs were stimulated for 24 or 48 h, fixed with 4% paraformaldehyde (pFA) and stained with PE-conjugated anti-CD80 (1:12.5, 557227, BD pharmingen), allophycocyanin-conjugated CD83 (1:25, 551073, BD Pharmingen), FITC-conjugated anti-CD86 (1:25, 555657, BD Pharmingen), PE-conjugated anti-HLA-DR (1:25, 555812, BD Pharmingen), or PE-Cy7-conjugated anti-CD40 (1:100, 2165055, Sony Biotechnology). Expression levels after RNA interference were determined using anti-DDX3 or anti-MAVS (1:50, 2635S or 3993S, Cell Signaling) followed by PE-conjugated donkey anti-rabbit (1:200, Jackson Immuno Research). HIV-1 infection levels were assessed using anti-p24 (1:200, KC57-RD1, Beckman Coulter). Flow cytometric analysis was performed using the FACSCanto II (BD Biosciences) and FlowJo software v10.

Stunnenberg M., Sprokholt J.K., van Hamme J.L., Kaptein T.M., Zijlstra-Willems E.M., Gringhuis S.I, & Geijtenbeek T.B. (2020). Synthetic Abortive HIV-1 RNAs Induce Potent Antiviral Immunity. Frontiers in Immunology, 11, 8.

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Measuring SIV Env-specific Plasma IgG Binding

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Indirect surface staining was used to evaluate the ability of SIV Env-specific plasma IgG from AGMs and RMs to bind the surface of CEM.NKR_CCR5 CD4⁺ T cells [54 (link)] infected with SIVsab92018ivTF or SIVmac251. Infections with replication competent infectious molecular clone virus (IMC) were performed using DEAE-Dextran as described previously [51 (link), 55 (link)]. At 48 h postinfection, the infected CEM.NKR_CCR5 cells were incubated with a 1:100 dilution of plasma for 2 h at 37 °C and then stained with a vital dye (Live/Dead Fixable Aqua Dead Cell Stain; Invitrogen) to exclude nonviable cells from subsequent analyses. Primary Ab binding was detected by secondary labeling with fluorescein isothiocyanate (FITC)-conjugated goat anti-rhesus IgG (SouthernBiotech Inc., Birmingham, AL), and SIV-infected cells were identified by staining for intracellular expression of p24/27 (KC57-RD1; Beckman Coulter) using standard methods. Binding was quantified as % of live, p24/27 positive, FITC positive cells after subtracting the background binding of week 0 samples. Mock-infected cells were used to establish negative gates.

Nguyen Q.N., Martinez D.R., Himes J.E., Whitney Edwards R., Han Q., Kumar A., Mangan R., Nicely N.I., Xie G., Vandergrift N., Shen X., Pollara J, & Permar S.R. (2018). Predominant envelope variable loop 2-specific and gp120-specific antibody-dependent cellular cytotoxicity antibody responses in acutely SIV-infected African green monkeys. Retrovirology, 15, 24.

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Knockdown of Autophagy Receptors and Viral Antigen Expression

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HeLa‐CIITA cells were incubated in 6‐well plates using 2–4.10⁵ cells/well using OPTIMEM (Gibco) complemented with 10% FBS and 1% penicillin/streptomycin. Twenty‐four hours later, cells were transfected with 40 pmol of siRNA targeting NDP52 (L‐010637‐00‐0005), OPTN (L‐016269‐00‐0005), p62 (L‐010230‐00‐0005), T6BP (L‐016892‐00‐0020 Dharmacon or SI02781296, Qiagen), CANX (SI02663367 and SI02757300, Qiagen) or a scrambled siRNA as control (D‐001810‐10‐20, Dharmacon), using Lipofectamine RNAiMax (13778–150, Thermo Fisher) as transfection reagent. After 24 h of transfection, cells were transfected with the cDNA encoding the viral antigens (1 μg per well of a 6‐well plate) using Viromer RED (Lipocalyx) and following manufacturer instructions. Twenty‐four hours later, Gag and pp65 expressions were assessed using anti‐Gag antibody (KC57‐RD1, Beckman‐Coulter) and anti‐pp65 antibody (mouse, Argene) combined with goat anti‐mouse antibody (AF488, Thermo Fisher), respectively.

Sarango G., Richetta C., Pereira M., Kumari A., Ghosh M., Bertrand L., Pionneau C., Le Gall M., Grégoire S., Jeger‐Madiot R., Rosoy E., Subra F., Delelis O., Faure M., Esclatine A., Graff‐Dubois S., Stevanović S., Manoury B., Ramirez B.C, & Moris A. (2022). The Autophagy Receptor TAX1BP1 (T6BP) improves antigen presentation by MHC‐II molecules. EMBO Reports, 23(12), e55470.

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Analyzing Breg-Mediated Inhibition of CD4+ T Cells

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Proliferation dye VPD450 (BD Bioscience)-labeled total or Breg-depleted PBMCs from HIV⁺ individuals were stimulated with HIV-peptide (NIH AIDS repository) pool spanning nef, env, gag and pol (2 ug/ml each) or 500 µM SAHA (Sigma). After 96 hours the frequencies of either CD8⁺CD107a⁺ (cytotoxic CD8⁺ T cells), infected CD4⁺ T cells (using KC57-Rd1, Beckman-Coulter, antibody that binds HIV-1 proteins 55, 39 33 and 24 KDa core) and proliferation of CD4+ T cells were determined by flow-cytometry. To determine the effect of PD-L1/PD-1 and IL-10/IL-10R interactions in Breg-mediated inhibition of CD4+ T cell proliferation, VPD450-labeled FACS-purified CD4⁺ T cells were activated using Anti-Biotin MACSiBead loaded CD2, CD3, and CD28 antibodies (Miltenyi, T Cell Activation/Expansion Kit) and IL-2 (20U, NIH AIDS repository), cultured with medium alone, with Bregs, with Bregs and IL-10R blocking antibody (20 ug/ml, BD Pharmingen), with Bregs and PD-1 blocking antibody (10 ug/ml, BD Pharmingen) or with Bregs with both IL-10R and PD-1 blocking antibodies. After 72 hours, proliferation was determined by flow cytometry.

Siewe B., Wallace J., Rygielski S., Stapleton J.T., Martin J., Deeks S.G, & Landay A. (2014). Regulatory B Cells Inhibit Cytotoxic T Lymphocyte (CTL) Activity and Elimination of Infected CD4 T Cells after In Vitro Reactivation of HIV Latent Reservoirs. PLoS ONE, 9(4), e92934.

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Flow Cytometry Analysis of HIV Gag Protein

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For flow cytometry analysis and sorting, Jurkat or primary T cells were suspended in phosphate buffered saline (PBS) containing 1% FBS (FACS buffer). Dead cells were excluded in all analyses and sorting experiments using propidium iodide (PI). Intracellular Gag staining was carried out using a Gag monoclonal antibody conjugated to Phycoerythrin (KC-57 RD1 Beckman Coulter). 1x10⁵ cells from a HIV GPV- zip coded library were washed once with FACS buffer and fixed with 100 μl of BD cytofix for 10 minutes at room temperature in the dark. Cells were then washed twice with FACS buffer then once with BD perm/wash buffer. Staining was carried out at a 1:200 dilution of antibody in 1x BD perm buffer. The cells were incubated in the dark at room temperature for 15 minutes, washed twice, then resuspended in 200 μl FACS buffer. Acquisition was carried out on the FITC channel for GFP and PE channel for Gag. Cell fluorescence was assessed using FACSCanto II (BD Biosciences) and data were analyzed using FlowJo software, version 9.9 (FlowJo, LLC., Ashland, Oregon).

Read D.F., Atindaana E., Pyaram K., Yang F., Emery S., Cheong A., Nakama K.R., Burnett C., Larragoite E.T., Battivelli E., Verdin E., Planelles V., Chang C.H., Telesnitsky A, & Kidd J.M. (2019). Stable integrant-specific differences in bimodal HIV-1 expression patterns revealed by high-throughput analysis. PLoS Pathogens, 15(10), e1007903.

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Quantifying HIV-1 Protein Expression Levels

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To quantify HIV-1 protein expression levels for different RNA species in the HIV-1Δenv-d2GFP A7 mutants as compared to RNA levels found in WT HIV-1Δenv-d2GFP, direct immunofluorescence by flow cytometry was used. At 20 hours after TNF-alpha induction, cells were fixed with a final of 2% formaldehyde (Tousimis. cat#1008A) for 15 min at room temperature. Cells were then centrifuged and supernatant aspirated. Cells were permeabilized with 1:1 methanol:acetone and incubated on ice for 10 min. Cells were then washed with Stain Buffer (DPBS without calcium and magnesium, 2% FBS, 2 mM EDTA, 0.1% IGEPAL CA-630) with centrifugation and supernatant aspiration between washes. Cells were centrifuged and supernatant aspirated and resuspended in 50 μL of Stain Buffer and 1 μL of KC57-RD1 (Beckman Coulter. cat#6604667) and incubated at room temperature in the dark for 15 minutes. Cells were then washed with 1 mL of Flow Buffer (DPBS without calcium and magnesium, 2% FBS, 2 mM EDTA) and centrifuged and supernatant aspirated. Cells were then resuspended in 200 μL of Flow Buffer. Fluorescence was detected using a LRSII flow cytometer (BD Biosciences) excitation at 531 nm, filtered at 585/42 nm, and recorded at 568–590 nm.

Hansen M.M., Wen W.Y., Ingerman E., Razooky B.S., Thompson C.E., Dar R.D., Chin C., Simpson M.L, & Weinberger L.S. (2018). A Post-Transcriptional Feedback Mechanism for Noise Suppression and Fate Stabilization. Cell, 173(7), 1609-1621.e15.

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Flow Cytometric Analysis of Intracellular p24

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For intracellular p24 staining, U1 cells were washed with PBS containing 10% human serum followed by fixation and permeabilization using a fixation/permeabilization kit (eBiosciences). Permeabilized cells were then incubated with 100 μl of a 1:100 dilution of phycoerythrin (PE)-conjugated mouse anti-p24 monoclonal antibody (MAb) (KC57-RD1; Beckman Coulter, Inc.) for 30 min at 4°C with intermittent mixing. After the samples were washed twice, the cells were analyzed by flow cytometry.

Tyagi P., Pal V.K., Agrawal R., Singh S., Srinivasan S, & Singh A. (2020). Mycobacterium tuberculosis Reactivates HIV-1 via Exosome-Mediated Resetting of Cellular Redox Potential and Bioenergetics. mBio, 11(2), e03293-19.

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