Ack lysing buffer

Manufactured by Merck Group

Sourced in Germany

ACK lysing buffer is a reagent used for the lysis of red blood cells (erythrocytes) in cell samples. It is a commonly used solution in various laboratory techniques, such as flow cytometry and cell isolation procedures. The buffer works by disrupting the cell membranes of red blood cells, allowing for the selective removal of these cells from the sample.

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Lab products found in correlation

Percoll density gradient media, by Merck Group (3 mentions) Collagenase 4, by Merck Group (3 mentions) Hyaluronidase, by Merck Group (3 mentions) Cytof system, by Standard BioTools (2 mentions) Dnaase, by Merck Group (2 mentions) Cryotubes, by Greiner (2 mentions) Edta vacutainer, by BD (2 mentions) Fetal bovine serum (fbs), by Capricorn (2 mentions) Leucosep tube, by Greiner (2 mentions) Phosphate buffered saline (pbs), by Merck Group (2 mentions) Fix perm buffer, by Standard BioTools (1 mentions) Dnase 1, by Merck Group (1 mentions) Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Gibco penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Live dead aqua, by Thermo Fisher Scientific (1 mentions) Mhcii, by BioLegend (1 mentions) Cd103, by BioLegend (1 mentions) Gm csf, by Thermo Fisher Scientific (1 mentions) Complete rpmi, by Corning (1 mentions) 24 well plate, by Corning (1 mentions)

15 protocols using ack lysing buffer

Comprehensive Kidney Immune Profiling

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All the kidney samples were processed in the same batch. CyTOF analysis was performed by PLTTech, Inc. (Hangzhou, China) according to a published protocol [12 (link)]. The kidney tissue was dissociated into a single-cell suspension with a mixture of DNase I, collagenase IV, and hyaluronidase (Sigma-Aldrich). Immune cells were enriched using Percoll density gradient media (Sigma-Aldrich), and erythrocytes were fully removed using ACK Lysing Buffer (Sigma-Aldrich). Qualified samples were blocked and stained with a mixed panel of surface antibodies, followed by overnight fixation. After fixing with Fix & Perm Buffer (Fluidigm), the cells were incubated in an intracellular antibody mix. The signals of the stained cells were detected using a CyTOF system (Fluidigm). The types of immune cells were identified via nonlinear dimensionality reduction (t-SNE), followed by density clustering.

Zhang W., Li L., Zhang T., Gao X., Wang Z., Ming S., Fang Z., Liu M., Dong H., Zhu B., Liao J., Zeng J., Peng Y, & Gao X. (2021). An Immune Atlas of Nephrolithiasis: Single-Cell Mass Cytometry on SIRT3 Knockout and Calcium Oxalate-Induced Renal Injury. Journal of Immunology Research, 2021, 1260140.

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Spleen Cell Isolation and Preparation

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The spleen samples collected in RPMI medium were homogenised and passed through 70 μm cell mesh strainers (BD Biosciences, UK). Cells were washed in RPMI medium by centrifugation and red blood cells were lysed with ACK lysing buffer (Sigma-Aldrich, UK). Following lysis, cells were washed twice in RPMI by centrifugation and re-suspended in RPMI complete medium (RPMI with 10% foetal bovine serum (Gibco, UK), 1% Gibco penicillin-streptomycin (10,000 U/ml) (Life Technologies, UK) and 1% Gibco MEM non-essential amino acids (100X) (Life Technologies, UK)), counted and stored at 4° C overnight prior to flow cytometric analysis.

Gordon L., Mabbott N., Wells J., Kulik L., Juleff N., Charleston B, & Perez-Martin E. (2022). Foot-and-mouth disease virus localisation on follicular dendritic cells and sustained induction of neutralising antibodies is dependent on binding to complement receptors (CR2/CR1). PLoS Pathogens, 18(5), e1009942.

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Multiparametric Immune Cell Profiling

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Spleens, mesenteric lymph nodes, and Peyer’s patches were removed and forced through 70 μM cell strainers to create single cell suspensions. Red blood cells in splenocyte suspensions were lysed with ammonium chloride potassium (ACK) lysing buffer (Sigma). Dead cells were stained with live/dead Aqua (Life Technologies). Surface antigens were stained with the following antibodies: CD11c (eBioscience, clone N418), MHCII (BioLegend, clone M5/114.15.2), CD3ε (BioLegend, clone 145-2C11), CD19 (BD biosciences, clone 1D3), CD8α (BD biosciences, clone 53–6.7), CD4 (BioLegend, clone RM4-5), CD40, CD80, CD86, MHCI-K^b (BD biosciences, clone AF6-88.5), CD44 (BD biosciences, clone IM7), PD-1 (BioLegend, clone RMP1-14), CD103 (BioLegend, clone 2E7), Ly6C (BioLegend, clone HK1.4), NKp46 (BioLegend, clone 29A1.4), IFNAR1 (BioLegend, clone MAR1-5A3), KLRG1 (Tonbo, clone 2F1), CD62L (BioLegend, clone MEF-14), SIINFEKL peptide in grove antibody (BioLegend, clone 25-D1.16), CD45.1 (BioLegend clone A20), CD45.2 (BioLegend clone 104). MHCI K^b tetramers with the CW3 immunodominant epitope SWVPRLYQL were generated as described [21 (link)].

Nice T.J., Osborne L.C., Tomov V.T., Artis D., Wherry E.J, & Virgin H.W. (2016). Type I Interferon Receptor Deficiency in Dendritic Cells Facilitates Systemic Murine Norovirus Persistence Despite Enhanced Adaptive Immunity. PLoS Pathogens, 12(6), e1005684.

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Comprehensive CyTOF Immune Profiling of Tumor Samples

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CyTOF analyses were performed by PLTTech Inc. (Hangzhou, China) according to the previously described protocol.11 (link) In brief, tumour tissue was dissociated into single cells with DNAase, collagenase IV and hyaluronidase (Sigma-Aldrich, Saint Louis, MO, USA). Immune cells were enriched using Percoll density gradient media (Sigma-Aldrich), and red blood cells were removed using ACK Lysing Buffer (Sigma-Aldrich). Qualified samples were blocked and stained for 30 min with a surface antibody mix panel developed in-house (online table S1), followed by fixation overnight. Permeabilization buffer was applied, and the cells were incubated in an intracellular antibody mix. The cells were rinsed, and the signals were detected using a CyTOF system (Helios, Fluidigm, South San Francisco, CA, USA). The types of immune cells were identified via nonlinear dimensionality reduction [t-distributed stochastic neighbour embedding (tSNE)], followed by density clustering.12 (link)

Zhang Q., Lou Y., Yang J., Wang J., Feng J., Zhao Y., Wang L., Huang X., Fu Q., Ye M., Zhang X., Chen Y., Ma C., Ge H., Wang J., Wu J., Wei T., Chen Q., Wu J., Yu C., Xiao Y., Feng X., Guo G., Liang T, & Bai X. (2019). Integrated multiomic analysis reveals comprehensive tumour heterogeneity and novel immunophenotypic classification in hepatocellular carcinomas. Gut, 68(11), 2019-2031.

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Generation of GBM-associated MDSCs

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GBM-associated MDSCs were generated as in previous reports (13 (link),15 (link)). Bone marrow (BM) cells from C57BL/6 mouse were flushed out from femurs with complete RPMI using a 10 mL syringe and 25-gauge needle into complete RPMI (Corning). BM cells were centrifuged (10 minutes, 1500 RPM, at 4°C), and red blood cells were lysed using ACK lysing buffer (Sigma) for 5 minutes at RT. Cells were washed (5 minutes, 1500 RPM, at 4°C) with complete RPMI, counted, and plated into 24-well plates (Corning) at a density of 2.5×10⁵ cells per well with 50% complete RPMI, 50% conditioned media (8mL DMEM (Corning) supernatant that was collected from original seeding of 2×10⁶ CT2A cells after 72 hours of culture), and GM-CSF (40 ng/mL; PeproTech). After 3 days of culture, old media was removed by aspiration and new media (same as aforementioned) was added to the cells for an additional 3 days of incubation. Six days after BM isolation, cells were lifted by pipetting up and down, washed (10 minutes, 300 x g, at 4°C), and stained for characteristic flow cytometric analysis (CD11b⁺Ly6C⁺PD-L1⁺arginase-1⁺, Supplementary Table S3 and further functional assays as described below.

Lee-Chang C., Rashidi A., Miska J., Zhang P., Pituch K.C., Hou D., Xiao T., Fischietti M., Kang S.J., Appin C.L., Horbinski C., Platanias L.C., Lopez-Rosas A., Han Y., Balyasnikova I.V, & Lesniak M.S. (2019). Myeloid-derived suppressive cells promote B cell–mediated immunosuppression via transfer of PD-L1 in glioblastoma. Cancer immunology research, 7(12), 1928-1943.

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Isolation of Tumor-Associated Macrophages

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Briefly, FVB/N mice received subcutaneous injections of 5 × 10⁶ NT2.5 cells into the right upper mammary fat pad. One week post-tumor challenge, mice were vaccinated with 3 × 10⁶ irradiated 3T3neuGM cells or 3T3GM cells. Sorafenib (30 mg/kg) or vehicle was given by daily gavage starting on the day of vaccination. Tumors were harvested Day 12 post-treatment and suspended in 5 ml RPMI 1640 containing Liberase TM (Roche). Tumors were minced and incubated at 37 °C for 30 min. Suspensions were then passed through a 100-μm mesh nylon cell strainer (BD Falcon) to obtain single cell suspensions. Single cell suspensions were incubated with in ACK lysing buffer (Sigma) for 2 min at room temperature and washed twice in FACS-staining buffer (PBS, 2% heat-inactivated FCS). Pellets were resuspended in FACs staining buffer containing Fc Block (MACs Miltenyi Biotec) and incubated on at 4 °C for 20 min. CD11b⁺ TAMs were obtained through positive isolation (MACs Miltenyi Biotech). CD11b⁺ TAMs were incubated with FACs buffer containing biotinylated anti-F480 (eBioscience) and isolated by positive selection (MACs Miltenyi Biotec).

Sunay M.M., Foote J.B., Leatherman J.M., Edwards J.P., Armstrong T.D., Nirschl C.J., Hicks J, & Emens L.A. (2017). Sorafenib combined with HER-2 targeted vaccination can promote effective T cell immunity in vivo. International immunopharmacology, 46, 112-123.

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PBMC Stimulation with C. mast Lysate

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Research was in compliance with guidelines of the National Institutes of Health Institutional Review Board (Protocol #6145) and all procedures conformed to the tenets of the Declaration of Helsinki. Healthy human PBMCs were received by the NIH blood bank. Red bloods cells (RBCs) were separated using centrifugation (2000g × 15 min. at 25°C) and the ACCUSPIN™ System-Histopaque^®-1077. Remaining RBCs were lysed after a 1 min incubation at room temperature (RT) with ACK lysing buffer (Sigma). 5 × 10⁵ Cells were plated in 96 well round bottom plate in 200 μl of DMEM + 10% FBS. In addition cells were stimulated with 10μg of C. mast lysate for 72 hours.

Leger A.J., Desai J.V., Drummond R.A., Kugadas A., Almaghrabi F., Silver P., Raychaudhuri K., Gadjeva M., Iwakura Y., Lionakis M.S, & Caspi R.R. (2017). An ocular commensal protects against corneal infection by driving an Interleukin 17 response from mucosal γδ T cells. Immunity, 47(1), 148-158.e5.

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Isolation of Immune Cells from Rat Tissues

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Peripheral blood mononuclear cells (PBMCs) were isolated from tail vein blood using a Vacutainer (Fisher Scientific). Blood was centrifuged (5,000 rpm), the pellet was resuspended in 1 mL ammonium–chloride–potassium (ACK) lysing buffer (Life Technologies; 3‐5 min at room temperature [RT]), diluted in 10 mL PBS, and centrifuged (5 min, 1,800 rpm). The pellet was resuspended in R10: Roswell Park Memorial Institute (Sigma), 10% fetal bovine serum (Sigma), 1% L‐glutamine (Sigma), and 1% penicillin/streptomycin (Sigma).
Splenocytes were isolated from rat spleens by disruption through a 70‐µm cell strainer and centrifuged (1,200 rpm, 5 min, 4°C), and the pellet was resuspended in 3 mL ACK lysing buffer (2 minutes, RT). PBS was added to 25 mL, centrifugation was performed as before, and the pellet was resuspended in R10.
Liver‐infiltrating lymphocytes were obtained by perfusion of the liver with PBS and disruption through a 100‐µm then a 50‐µm cell strainer. Cells were centrifuged (5 minutes, 1,500 rpm), and the pellet was resuspended in 40% Percoll (GE). An underlay of 70% Percoll was applied, and samples were centrifuged (2,000 rpm, 25 minutes without brake). Fat and debris were removed and interphase was collected, diluted in PBS, and centrifuged (1,500 rpm, 5 minutes). The pellet was resuspended in 3 mL ACK and treated as for splenocytes.

Atcheson E., Li W., Bliss C.M., Chinnakannan S., Heim K., Sharpe H., Hutchings C., Dietrich I., Nguyen D., Kapoor A., Jarvis M.A., Klenerman P., Barnes E, & Simmonds P. (2019). Use of an Outbred Rat Hepacivirus Challenge Model for Design and Evaluation of Efficacy of Different Immunization Strategies for Hepatitis C Virus. Hepatology (Baltimore, Md.), 71(3), 794-807.

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Murine Splenocyte Immunophenotyping

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Individual spleens were removed and mechanically dissociated using a syringe plunger above 70 μm-pore size Falcon cell strainer (BD Biosciences). Red blood cells were lysed using ACK lysing buffer (Sigma). Single-cell suspensions were counted and incubated with anti-Fcγ III/II (CD16/32) receptor Ab (2.4G2) in PBS containing 3% FCS for 15 min, and immunolabelled for 30 min at 4 °C in the dark with the following fluorochrome-conjugated antibodies: PE-Cy7 anti-mouse CD8 (53–6.7), PerCP-Cy5.5 anti-CD3 (145-2c11), APC-H7 anti-mouse CD4 (GK1.5), APC anti-mouse B220 (RA3-6B2), BB515 anti-mouse CD62L (MEL14), APC anti-mouse CD44 (IM7) and/or PE anti-mouse CD25 (7D4). For Treg cells analyses, cells were fixed and permeabilized, after staining for surface markers, with eBioscience™ Foxp3/Transcription Factor Staining Buffer Set according to the manufacturer instructions and incubated with the antibody Alexa Flour 647 anti-Foxp3 (R16715). All antibodies were from BD Biosciences. Data were collected using FACSDiva software on a FACSCANTO II flow cytometer (BD Biosciences), and analysed using FlowJo software 10.0 (BD Biosciences).

de Sousa L.P., Ribeiro-Gomes F.L., de Almeida R.F., Souza T.M., Werneck G.L., Souza D.O, & Daniel-Ribeiro C.T. (2021). Immune system challenge improves recognition memory and reverses malaria-induced cognitive impairment in mice. Scientific Reports, 11, 14857.

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Isolating BWF1 Mouse Spleen Cells

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Spleen cells were isolated from 8-10-week-old male and female BWF1 mice and single cell suspensions prepared by passing cells through cell strainers (Fisher). ACK lysing buffer (Sigma, St Louis, MO, USA) was used to lyse and remove red blood cells. White blood cells were lysed with TRIzol (Invitrogen Inc., Carlsbad, CA) and RNA isolated as per the manufacturer’s protocol. Real-time PCR was performed with 100 ng of RNA from each sample with rodent IFI202b-specific primers and probe (Applied Biosystems, Foster City, CA, USA). All values were normalized to GAPDH levels.

Singh R.P., Hahn B.H, & Bischoff D.S. (2021). Interferon Genes Are Influenced by 17β-Estradiol in SLE. Frontiers in Immunology, 12, 725325.

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