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Ack solution

Manufactured by Thermo Fisher Scientific
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ACK solution is a specialized buffer used to lyse red blood cells in cell samples. It functions to selectively remove erythrocytes, allowing for the isolation and analysis of other cell types.

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Lab products found in correlation

Dnase 1, by Roche (4 mentions) Anti cd8 fitc, by BioLegend (3 mentions) Anti cd45 pacific blue, by BioLegend (3 mentions) Anti cd4 pe, by BioLegend (3 mentions) Anti ter119 fitc, by BioLegend (3 mentions) Anti sca1 pacific blue, by BioLegend (3 mentions) Anti pdgfrα pe, by BioLegend (3 mentions) Anti cd45 fitc, by BioLegend (3 mentions) Anti cd31 fitc, by BioLegend (3 mentions) Anti cd3 apc, by BioLegend (3 mentions) Liberase, by Roche (3 mentions) 70 m nylon mesh, by Corning (3 mentions) Anti nk1.1 apc cy7, by BioLegend (2 mentions) Anti b220 apc cy7, by BioLegend (2 mentions) Facsaria 2, by BD (2 mentions) Facsaria 3, by BD (2 mentions) Lsrii fortessa, by BD (2 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) Cell staining buffer, by BioLegend (1 mentions) Anti granzyme b, by BioLegend (1 mentions)

Spelling variants of this product

ACK lysis solution (9 citations) ACK lysing solution (4 citations)

18 protocols using ack solution

1

Multicolor Flow Cytometry of Tumors

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Cells were analyzed using LSRII (BD, Pharmingen) and sorted using FACS-ARIA II (BD, Pharmingen). The following antibodies were used: anti-CD45 FITC, anti-CD31 FITC, anti-Ter119 FITC, anti-Sca1 Pacific Blue, anti-PDGFRα PE, anti-CD3 APC, anti-CD8 FITC, anti-CD45 Pacific Blue, anti-CD4 PE, anti-NK1.1 APC-cy7, anti-B220 APC-cy7 (all purchased from Biolegend). Tumors were digested to single cell suspension enzymatically and filtered twice through 70 μm filters. Red blood cells were lysed with ACK solution (Gibco), washed twice with PBS, and then stained with the fluorophore-conjugated antibodies for 15 min at room temperature. The excess of unbound antibodies was washed out before acquisition in flow cytometry.
Tessaro F.H., Ko E.Y., De Simone M., Piras R., Broz M.T., Goodridge H.S., Balzer B., Shiao S.L, & Guarnerio J. (2022). Single-cell RNA-seq of a soft-tissue sarcoma model reveals the critical role of tumor-expressed MIF in shaping macrophage heterogeneity. Cell reports, 39(12), 110977.
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2

Multiparametric Flow Cytometry Analysis of Tumor Immune Microenvironment

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Tumors were digested to single cell suspension enzymatically and filtered twice through 70μm filters. Red blood cells were lysed with ACK solution (Gibco), washed twice with Cell Staining Buffer (BioLegend), and then stained with the fluorophore-conjugated antibodies for 30 min at 4 °C. The excess of unbound antibodies was washed out before acquisition in flow cytometry. Cells were analyzed using ID700 Spectral Cell Analyzer (SONY) and sorted using FACS-ARIA III (BD, Pharmingen). The following antibodies were for flow cytometry: anti-CD45 PAC (30-F11), anti-CD8b APC-Cy7 (YTS156.7.7), anti-CD4 PE(H129.19), anti-NK1.1 APC-Cy7 (PK136), anti-CD11b APC (M1/70), anti-Ly6c PE (HK1.4), anti F4/80 (BM8), anti-CXCR6 APC (SA051D1), anti-GranzymeB, anti-CD90 FITC (30-H12), anti-CD73 APC (TY/11.8), anti-PD-1 APC (RMP1-30) all purchased from BioLegend and used at 1:100 dilution. Anti-CD31-PE (eBioscience, 390, 1:100), anti-GLUT1 (Novus, Fgi.72, 1:100), anti-Cxcl16 (Bioss, BS-1441R, 1:50) and anti-Rabbit IgG AF488 (Invitrogen, A21245, 1:100) were used to identify CAF populations. Ghost Dye Red 710 (Tonbo, Cat #13-0871-T100) was used to exclude dead cells from the analysis.
Broz M.T., Ko E.Y., Ishaya K., Xiao J., De Simone M., Hoi X.P., Piras R., Gala B., Tessaro F.H., Karlstaedt A., Orsulic S., Lund A.W., Chan K.S, & Guarnerio J. (2024). Metabolic targeting of cancer associated fibroblasts overcomes T-cell exclusion and chemoresistance in soft-tissue sarcomas. Nature Communications, 15, 2498.
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3

Multicolor Flow Cytometry of Tumors

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Cells were analyzed using LSRII (BD, Pharmingen) and sorted using FACS-ARIA II (BD, Pharmingen). The following antibodies were used: anti-CD45 FITC, anti-CD31 FITC, anti-Ter119 FITC, anti-Sca1 Pacific Blue, anti-PDGFRα PE, anti-CD3 APC, anti-CD8 FITC, anti-CD45 Pacific Blue, anti-CD4 PE, anti-NK1.1 APC-cy7, anti-B220 APC-cy7 (all purchased from Biolegend). Tumors were digested to single cell suspension enzymatically and filtered twice through 70 μm filters. Red blood cells were lysed with ACK solution (Gibco), washed twice with PBS, and then stained with the fluorophore-conjugated antibodies for 15 min at room temperature. The excess of unbound antibodies was washed out before acquisition in flow cytometry.
Tessaro F.H., Ko E.Y., De Simone M., Piras R., Broz M.T., Goodridge H.S., Balzer B., Shiao S.L, & Guarnerio J. (2022). Single-cell RNA-seq of a soft-tissue sarcoma model reveals the critical role of tumor-expressed MIF in shaping macrophage heterogeneity. Cell reports, 39(12), 110977.
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4

Multiparameter Flow Cytometry Analysis

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Cells were analyzed using the LSR II (BD, Pharmingen) and sorted using FACSAria III (BD, Pharmingen), using FACSDiva software (BD, v8). Analysis was performed with FlowJo (v10). Samples were stained with the following antibodies at 1 ul antibody per 100ul cell suspension: anti-CD45 FITC, anti-CD31 FITC, anti-Ter119 FITC, anti-Sca1 Pacific Blue, anti-PDGFRα PE, anti-CD3 APC, anti-CD8 FITC, anti-CD45 Pacific Blue, anti-CD4 PE (all purchased from Biolegend). Tumors were enzymatically digested to single-cell suspension and filtered twice through 70 μm filters. Red blood cells were lysed with ACK solution (Gibco), washed twice with PBS, and then stained with the fluorophore-conjugated antibodies for 15 minutes at room temperature. The excess of unbound antibodies was washed out before acquisition in flow cytometry. Intracellular staining was performed to detect dsRNAs. Briefly, cells were fixed and permeabilized by using Cyto-Fast fix/perm buffers (Biolegend), following vendor’s protocol. After performing the staining for the surface markers, J2 antibody (Jena Bioscience, 2ug total) was added to the cells and incubated for 1 hour at room temperature, followed by incubation for 20 minutes with Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) secondary antibody (A32723, Invitrogen/Thermo Fisher).
Piras R., Ko E.Y., Barrett C., De Simone M., Lin X., Broz M.T., Tessaro F.H., Castillo-Martin M., Cordon-Cardo C., Goodridge H.S., Di Vizio D., Batish M., Lawrenson K., Chen Y.G., Chan K.S, & Guarnerio J. (2022). circCsnk1g3- and circAnkib1-regulated interferon responses in sarcoma promote tumorigenesis by shaping the immune microenvironment. Nature Communications, 13, 7243.
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5

Biopsy and Tissue Isolation for PCL Implants

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To harvest biopsies of PCL implants for analysis, mice were anesthetized with isoflurane before an incision was made over the surface of the implant. The implant and any adherent encapsulating tissue were pulled through the incision and excised and the incision was closed with sutures. Tissues for RNA were flash frozen in isopentane on dry ice and stored at −80 °C until analysis. Tissues for flow cytometry were placed into PBS and stored on ice, and tissues for histology were immediately placed in 4% paraformaldehyde. For blood isolation, mice were anesthetized before intracardiac blood draw with EDTA. RBCs were lysed in an ACK solution (Gibco) before washing in PBS. Pellets were resuspended in Trizol and stored at −80 °C until analysis.
Morris A.H., Hughes K.R., Oakes R.S., Cai M.M., Miller S.D., Irani D.N, & Shea L.D. (2020). Engineered immunological niches to monitor disease activity and treatment efficacy in relapsing multiple sclerosis. Nature Communications, 11, 3871.
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6

Isolation of Lung Leukocytes

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Lung tissues were harvested after perfusion and disrupted into small pieces and then were digested with Liberase TM (Roche) plus DNase I (Roche) at 37 °C for 20 min. Tissue pieces were strained into single cells, and the leukocytes were purified by centrifugation using 40% Percoll (GE Healthcare Life Sciences) in PBS solution, followed by ACK solution (Life technologies) treatment.
Huang Y., Guo L., Qiu J., Chen X., Hu-Li J., Siebenlist U., Williamson P.R., Urban JF J.r, & Paul W.E. (2014). IL-25-responsive, lineage-negative KLRG1hi cells are multipotential “inflammatory” type 2 innate lymphoid cells. Nature immunology, 16(2), 161-169.
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7

CD4 T-cell Isolation and Adoptive Transfer

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Spleens and lymph nodes were harvested, homogenized, and RBC were lysed by ACK solution (Life Technologies Carlsbad, CA) as previously described [29] (link). CD4 T-cells were purified using Dynal magnetic beads according to the manufacturer’s protocol (Invitrogen Dynal, Norway). CD4 cells were resuspended in 0.2 ml PBS prior to transfer; unless otherwise noted 1E6 cells per mouse were adoptively transferred via retro-orbital injection.
Durham N.M., Nirschl C.J., Jackson C.M., Elias J., Kochel C.M., Anders R.A, & Drake C.G. (2014). Lymphocyte Activation Gene 3 (LAG-3) Modulates the Ability of CD4 T-cells to Be Suppressed In Vivo. PLoS ONE, 9(11), e109080.
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8

Isolation of Lung Leukocytes

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Lung tissues were harvested after perfusion and disrupted into small pieces and then were digested with Liberase TM (Roche) plus DNase I (Roche) at 37 °C for 20 min. Tissue pieces were strained into single cells, and the leukocytes were purified by centrifugation using 40% Percoll (GE Healthcare Life Sciences) in PBS solution, followed by ACK solution (Life technologies) treatment.
Huang Y., Guo L., Qiu J., Chen X., Hu-Li J., Siebenlist U., Williamson P.R., Urban JF J.r, & Paul W.E. (2014). IL-25-responsive, lineage-negative KLRG1hi cells are multipotential “inflammatory” type 2 innate lymphoid cells. Nature immunology, 16(2), 161-169.
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9

PBMC Isolation from Whole Blood

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Blood samples were processed within 4 h of collection. 20 mL of 2% FBS/PBS was added to 20 mL of whole blood. This was layered onto Ficoll-Paque. Sample was centrifuged at 1300× g for 20 min at the slowest acceleration and with break off. After centrifugation, the PBMC ring was removed using a pipette. The ring was topped up with 2% FBS/PBS and centrifuged again at 300× g. Any excess red blood cells were lysed with ACK solution (Life Technologies, A1049201, Waltham, MA, USA) and cells were washed again.
Sivakumar S., Abu-Shah E., Ahern D.J., Arbe-Barnes E.H., Jainarayanan A.K., Mangal N., Reddy S., Rendek A., Easton A., Kurz E., Silva M., Soonawalla Z., Heij L.R., Bashford-Rogers R., Middleton M.R, & Dustin M.L. (2021). Activated Regulatory T-Cells, Dysfunctional and Senescent T-Cells Hinder the Immunity in Pancreatic Cancer. Cancers, 13(8), 1776.
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10

Single-cell Isolation from Murine Tissues

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Lung tissues were collected after perfusion, disrupted into small pieces and digested for 20 min at 37 °C with Liberase (Roche) plus DNase I (Roche). Tissue pieces were strained into single cells followed by treatment with ACK solution (Life Technologies).
MLN tissues were collected and strained into single cells.
Small intestines were collected, and the contents were emptied. Peyer’s patches were removed, and small intestines were opened longitudinally, cut into small pieces and shaken at 37 °C for 40 min in PBS media containing 10% fetal bovine serum (FBS), 5 mM EDTA and 1 mM dithiothreitol to dissociate intraepithelial leukocytes. The remaining fragments were washed twice with PBS and digested at 37 °C for 45 min in HBSS media containing Liberase and DNase I. The digested tissues were strained to yield a single-cell suspension, which was followed by treatment with ACK solution.
Liver tissues were collected after perfusion and were strained into single cells. Hepatocytes were removed by centrifugation at 30 g for 3 min, and leukocytes in the supernatant were purified by centrifugation with 33% Percoll in PBS solution, followed by treatment with ACK solution.
BM cells were obtained by flushing tibias and femurs, followed by treatment with ACK solution.
Minion F.C., Adams C, & Hsu T. (2000). R1 Region of P97 Mediates Adherence of Mycoplasma hyopneumoniae to Swine Cilia. Infection and Immunity, 68(5), 3056-3060.
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