Clone 30 f11

Manufactured by BioLegend

Sourced in United States

The Clone 30-F11 is a fluorochrome-conjugated monoclonal antibody that recognizes the mouse CD45 antigen. CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of all nucleated hematopoietic cells.

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Lab products found in correlation

Clone 53 6, by BioLegend (6 mentions) Clone m1 70, by BioLegend (6 mentions) Clone n418, by BioLegend (5 mentions) Clone 145 2c11, by BioLegend (5 mentions) Clone gk1, by BioLegend (5 mentions) Clone 6d5, by BioLegend (4 mentions) Clone rb6 8c5, by BioLegend (4 mentions) Clone ra3 6b2, by BioLegend (4 mentions) Clone bm8, by BioLegend (3 mentions) Clone 1a8, by BioLegend (3 mentions) Facscanto 2, by BD (3 mentions) Clone pc61, by BioLegend (3 mentions) Anti cd45, by BioLegend (3 mentions) Apc cy7, by BioLegend (3 mentions) Clone rb6 8c5, by Cytek Biosciences (3 mentions) Clone m1 70, by BD (3 mentions) Cd11b, by BioLegend (2 mentions) Clone hk1, by BioLegend (2 mentions) Percoll, by GE Healthcare (2 mentions) Clone 17a2, by BioLegend (2 mentions)

Spelling variants of this product

Anti-CD45 (clone 30-F11, (27 citations) Anti-mouse CD45 (clone 30-F11, (9 citations) Anti-CD45 APC/Cy7 (clone 30-F11; (6 citations) CD45-APC/Cy7 (clone 30-F11, (5 citations) Anti-CD45-FITC (clone 30-F11, (5 citations) Antimouse CD45-APC (clone 30-F11; (3 citations) FITC clone 30-F11 (2 citations) Murine CD45-PE (Clone 30-F11) (2 citations) Rat anti‐CD45 clone 30‐F11 (2 citations) Clone 30-F11 Brilliant Violet 421 (2 citations)

48 protocols using clone 30 f11

Isolation and Purification of Brain Immune Cells

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Brain hemispheres were manually homogenized in 10 ml HBSS through a
70-μm cell strainer, centrifuged at 200g, 4 °C
for 5 min and resuspended in 800 μl 0.5% BSA in HBSS. Myelin depletion
(Miltenyi Biotec) was carried out according to the manufacturer’s
instructions. Cells were incubated with anti-mouse CD16/32 (5 ng
μl⁻¹, BioLegend, clone 93) for 10 min at 4
°C to block Fc receptor binding, and dead cells were identified using
LIVE⁄DEAD Fixable Dead Cell Stain Kit (ThermoFisher Scientific). Cells
were stained with antibodies in 100 μl PBS solution for 15 min at 4
°C. The following antibodies were used: CD45 (1 ng
μl⁻¹, Biolegend, clone 30F-11), CD11b (1 ng
μl⁻¹, Biolegend, clone M1/70) and Ly6G (1 ng
μl⁻¹, Biolegend, clone 1A8). Cells were sorted
using the BD Aria Fusion II (BD Biosciences) and stored in 300 μl RNA
later solution (ThermoFisher Scientific) at 4 °C. Between approximately
15,000 and 50,000 macrophages, neutrophils or microglia per ischemic hemisphere
were collected.

Liu Q., Johnson E.M., Lam R.K., Wang Q., Bo Ye H., Wilson E.N., Minhas P.S., Liu L., Swarovski M.S., Tran S., Wang J., Mehta S.S., Yang X., Rabinowitz J.D., Yang S.S., Shamloo M., Mueller C., James M.L, & Andreasson K.I. (2019). Peripheral TREM1 responses to brain and intestinal immunogens amplify stroke severity. Nature immunology, 20(8), 1023-1034.

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Multicolor Flow Cytometry for Immune Cell Profiling

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Approximately 10⁶ cells were suspended in 200 μl HBSS
buffer. Fc receptor binding was blocked with 100 μl solution of
anti-mouse CD16/CD32 (5 ng μl⁻¹, BioLegend, clone 93)
in PBS for 10 min at 4 °C followed by staining with 100 μl
solution of antibodies for 15 min at 4 °C. The following antibodies were
used for surface receptor detection: CD45 (1 ng μl⁻¹,
Biolegend, clone 30F11), CD11b (1 ng μl⁻¹, Biolegend,
clone M1/70), Ly6G (1 ng μl⁻¹, Biolegend, clone 1A8),
TREM1 (2.5 ng μl⁻¹, R&D, clone 174031) and isotype
control (rat IgG2A phycoerithrin (PE)-conjugated antibody, R&D, clone
54447). Cells were washed with HBSS buffer, resuspended in 200 μl of HBSS
buffer and analyzed with a LSR II cytometer (BD Biosciences) and analyzed using
FlowJo software (Tree Star Inc.). For
Cx3cr1^GFPCcr2^RFPflow cytometry, we applied CD11b PE-Cy7 and TREM1 PE: compensation controls for
GFP channel consisted of microglia derived from naïve
Cx3cr1^GFPCcr2^RFPbrain; for RFP controls were macrophage/monocytes derived from naïve
Cx3cr1^GFPCcr2^RFPblood.

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Cell Cycle Analysis of IL10-/- BMDMs

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Il10^-/- BMDMs were cultured for 16 hours with 500nM (+)-JQ1. Cells were washed with cold 1x PBS followed by staining with LIVE/DEAD Fixable Blue Dead Cell Stain Kit (1:1000; Invitrogen L23105). Cell cycle changes in (+)-JQ1 treated Il10^-/- BMDMs were assessed using the BD Pharmigen BrdU Flow kit according to the manufacturer (559619). Briefly, Il10^-/- BMDMs were co-cultured with BrdU and treated with (+)-JQ1 for 16 hours followed by fixation and permeabilization. Cells were treated with DNase followed by staining with FITC-conjugated anti-BrdU.
Bulk LPMCs from Il10^-/- mice were washed with cold 1x PBS and stained for viability followed by cell-surface marker staining for CD45 (1:100; Clone 30-F11, BioLegend), CD3ε (1:300; Clone 145-2C11, BioLegend), CD19 (1:200; Clone 6D5, BioLegend), CD11b (1:200; M1/70, BD Biosciences), CD11c (1:200; Clone N418, BioLegend), and F4/80 (1:200; Clone BM8, BioLegend) diluted in staining buffer (5% FBS/PBS). All samples were fixed with 4% PFA. Data was acquired with the FACSDIVA software using the BD LSR II and analyzed using FlowJo version 10.7.1.

Hoffner O’Connor M., Berglind A., Kennedy Ng M.M., Keith B.P., Lynch Z.J., Schaner M.R., Steinbach E.C., Herzog J., Trad O.K., Jeck W.R., Arthur J.C., Simon J.M., Sartor R.B., Furey T.S, & Sheikh S.Z. (2022). BET Protein Inhibition Regulates Macrophage Chromatin Accessibility and Microbiota-Dependent Colitis. Frontiers in Immunology, 13, 856966.

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Flow Cytometry and Immunohistochemistry Antibody Sources

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Sources of the antibodies for flow cytometry analysis are listed as follow: mouse Fc Block (1:100; 553142; BD Biosciences), mouse CD45 (1:100; clone 30-F11; Biolegend), mouse Gr1 (1:100; clone RB6-8C5; Biolegend), mouse CD11b (1:100; clone M1/70; eBiosciences), mouse CD11c (1:100; clone N418; Biolegend), mouse F4/80 (1:100; clone MB8; Biolegend), mouse Ly6G (1:100; clone 1A8; Biolegend), mouse Ly6C (1:100; clone HK1.4; Biolegend), human ENTPD2 (1:25; PA5-26333; Sigma-Aldrich), mouse Entpd2 (1:25; ab150503; Abcam), human ENTPD1 (1:100; clone A1; Biolegend), and mouse Entpd1 (1:100; clone Duha59; Biolegend). Sources of the antibodies for immunohistochemistry are listed as follow: human ENTPD2 (1:200; ab150503; Abcam), human GLUT1 (1:1000; ab15309Abcam), and human CA9 (1:500; ab1508L; Abcam).

Chiu D.K., Tse A.P., Xu I.M., Di Cui J., Lai R.K., Li L.L., Koh H.Y., Tsang F.H., Wei L.L., Wong C.M., Ng I.O, & Wong C.C. (2017). Hypoxia inducible factor HIF-1 promotes myeloid-derived suppressor cells accumulation through ENTPD2/CD39L1 in hepatocellular carcinoma. Nature Communications, 8, 517.

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Cytotoxicity Assay of T Cell-Mediated Cancer Cell Killing

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Co-culture of cancer cells and T cells was performed as previously described (66 (link)). Briefly, B16F10 cells were maintained in complete DMEM media (10% FBS and 50U/ml of Penicillin-Streptomycin). CD8 T cells isolated from spleen and lymph nodes from Pmel-1 or OT-I mice were stimulated with anti-CD3/CD28 beads (ThermoFisher, 11452D) and then cultured in complete RPMI 1640 media (10%FBS, 20mM HEPES, 1mM sodium pyruvate, 0.05mM 2-mercaptoethanol, 2mM L-glutamine, and 50U/ml streptomycin and penicillin, 20ng/ml recombinant mouse IL-2).
To test the sensitivity of cancer cells to T cell-driven cytotoxicity (OT-I or Pmel-1 model), we plated B16F10 cells (sgRosa26, sgTraf3, pEF1a-Empty, or pEF1a-Traf3) at equal density in all wells, and added T cells at ratios to cancer cells. With the OT-I model, we first incubated the B16F10 cells with 1nM SIINFEKL peptide for 2 hours prior to co-culture with T cells. With the Pmel-1 model, B16F10 cells were either pre-treated with 1ng/ml IFNγ overnight or untreated prior to the co-culture. There are 2–4 cell-culture replicates for each condition. After a one-day or three-day co-culture with T cells, we counted the remaining cancer cells by FACS using the precision count beads (BioLegend, 424902). T cells present in these cultures were gated out based on antibodies specific for CD45 (Biolegend, clone 30-F11) or CD8 (BioLegend, clone 53-6.7).

Gu S.S., Zhang W., Wang X., Jiang P., Traugh N., Li Z., Meyer C., Stewig B., Xie Y., Bu X., Manos M.P., Font-Tello A., Gjini E., Lako A., Lim K., Conway J., Tewari A.K., Zeng Z., Sahu A.D., Tokheim C., Weirather J.L., Fu J., Zhang Y., Kroger B., Liang J.H., Cejas P., Freeman G.J., Rodig S., Long H.W., Gewurz B.E., Hodi F.S., Brown M, & Liu X.S. (2021). Therapeutically increasing MHC-I expression potentiates immune checkpoint blockade. Cancer discovery, 11(6), 1524-1541.

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Spinal Cord Immune Cell Isolation

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Spinal cords were homogenized in ice cold calcium and magnesium free HBSS using a glass dounce homogenizer. Homogenates were passed through a 70 µm cell strainer and centrifuged for 5 min at 2000 rpm, 4 °C. Pellets were resuspended in HBSS and washed once more in HBSS. Pellets were then resuspended in 30% percoll (GE Healthcare) at 4 °C and then centrifuged for 10 min at 700 g with the brake off. The supernatant was aspirated and the cells were resuspended and blocked in flow cytometry staining buffer containing mouse Fc block (Biolegend) on ice for 10 min. Cells were centrifuged for 5 min at 300 g at 4 °C. Cells were stained with primary antibodies against CX3CR1 (1:100, Biolegend catalog # 149022 Clone SA011F11), Ly-6C (1:100, Biolegend catalog #128037 clone HK1.4), CD45 (1:200, Biolegend catalog # 103106 Clone 30-F11), Ly-6G (1:100, Biolegend catalog #565964 clone 1A8), and CD11b (1:200, Biolegend catalog #101262 clone M1/70) for 30 min at 4 °C in the dark. Cells were washed once and then stained for BrdU using the Phase-flow kit (Clone 3D4, Biolegend catalog # 370706) according to the manufacturer’s instructions. Stained cells were acquired on a BD LSRII flow cytometer and analyzed using the FlowJo software (see Supplementary Fig. 5B–D for gating strategy).

Hagan N., Kane J.L., Grover D., Woodworth L., Madore C., Saleh J., Sancho J., Liu J., Li Y., Proto J., Zelic M., Mahan A., Kothe M., Scholte A.A., Fitzgerald M., Gisevius B., Haghikia A., Butovsky O, & Ofengeim D. (2020). CSF1R signaling is a regulator of pathogenesis in progressive MS. Cell Death & Disease, 11(10), 904.

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Microglial Amyloid Uptake in 5xFAD Mice

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5xFAD mice (young adults: 10–12 weeks old; adults: 10–12 months old) were injected intraperitoneally with methoxy-XO4 (10 mg kg⁻¹ body weight, Tocris, cat. no. 4920). After 3 h, mice were transcardially perfused with ice-cold 1× PBS. Hippocampi were collected, and microglia were isolated by using density gradient separation and prepared as described previously with slight modifications^{10 (link)}. In addition to the microglia surface markers CD11b (1:200, clone M1/70, BioLegend, cat. no. 101212) and CD45 (1:200, clone 30-F11, BioLegend, cat. no. 103106), the following lineage markers were added: anti-CD3 (1:300, clone 17A2, BioLegend, cat. no. 100220), anti-CD19 (1:300, clone 6D5, BioLegend, cat. no. 115520), anti-CD45R (1:300, clone RA3-6B2, BD Biosciences, cat. no. 552772), Ly6C (1:300, clone AL-21, BD Biosciences, cat. no. 560593) and Ly6G (1:300, clone 1A8, BD Biosciences, cat. no. 560601) for 20 min at 4 °C. Percentage and MFI of methoxy-XO4-positive CD11b⁺CD45^low microglia were determined by flow cytometry using a FACSCanto II (BD Biosciences) and analyzed with FlowJo software (Tree Star).

d’Errico P., Ziegler-Waldkirch S., Aires V., Hoffmann P., Mezö C., Erny D., Monasor L.S., Liebscher S., Ravi V.M., Joseph K., Schnell O., Kierdorf K., Staszewski O., Tahirovic S., Prinz M, & Meyer-Luehmann M. (2021). Microglia contribute to the propagation of Aβ into unaffected brain tissue. Nature Neuroscience, 25(1), 20-25.

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Isolation and Characterization of Tumor-Associated Macrophages from Glioblastoma Xenografts

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Total TAMs (CD45⁺/Gr1⁻/CD11b⁺/DAPI⁻) were sorted from GBM xenografts through the fluorescence-activated cell sorting (FACS). Briefly, GBM xenografts were resected and mechanically dissociated and then digested in HBSS buffer containing DNase I (10 μg/mL, Sigma Aldrich, 10104159001) and Liberase (25 μg/mL, Roche, 5401020001) for 45 min at 37°C, and mixed by pipetting every 10 min. After digestion, cell suspensions were passed through a 70 μm filter, washed with cold PBS, and spun down (800 rpm) for 10 min at 4°C. Red blood cells in the samples were removed with the specific lysis buffer (BioLegend, 420301). The dissociated cells were re-suspended in RPMI 1640 medium and blocked with rat IgG (Santa Cruz, sc-2026) for 15 minutes before staining with specific antibodies for sorting. Antibodies used for FACS include: anti-CD45 (Biolegend, 103127, Clone 30-F11), anti-Gr1 (Biolegend, 108405, Clone RB6-8C5), and anti-CD11b (Biolegend, 101207, clone M1/70). The sorted TAMs (CD45⁺/Gr1⁻/CD11b⁺/DAPI⁻) were then used for RNA-seq analyses or the flow cytometric analyses to quantify macrophage phagocytosis of glioma cells in vivo as described below.

Zhai K., Huang Z., Huang Q., Tao W., Fang X., Zhang A., Li X., Stark G.R., Hamilton T.A, & Bao S. (2021). Pharmacological Inhibition of BACE1 Suppresses Glioblastoma Growth by Stimulating Macrophage Phagocytosis of Tumor Cells. Nature cancer, 2(11), 1136-1151.

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Characterizing Tumor Macrophage Subsets

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For in vivo macrophage analysis, tumor tissue was collected at set time points after tumor cell engraftment, digested, and filtered through a 70 μm strainer. Dissociated cells were further incubated with the following antibodies: anti-mouse CD16/CD32 (Multi Sciences, clone 2.4G2, Cat No. AM016-100), IgG2a, κ isotype ctrl (Biolegend, clone MOPC-173, Cat No. 400233), anti-mouse ABCA1 (BIO-RAD, clone 5A1-1422, Cat No. MCA2681), anti-mouse F4/80 (Biolegend, clone BM8, Cat No. 123110), anti-mouse\human CD11b CM1/70, Cat No. 101229), anti-mouse CD86 (Biolegend, clone GL-1, Cat No. 105005), anti-mouse CD206 (Biolegend, clone C068C2, Cat No. 141715), anti-mouse TNF-α (Biolegend, clone MP6-XT22, Cat No. 506303), anti-mouse IFN-γ (Biolegend, clone XMG1.2, Cat No. 506303), anti-mouse Arginase 1 (Abbexa, Polyclonal, Cat No. abx319179), and anti-mouse CD45 (Biolegend, clone 30-F11, Cat No. 103112). Intracellular staining was done using Fixation/Permeabilization kit (BD, 554722). Samples were subjected to FCM by using BD FACS Calibur, BD Aria I, and Beackman CytoFLEx. Data were analyzed with FlowJo (vX.0.7).

Wang S., Li L., Zuo S., Kong L., Wei J, & Dong J. (2022). Metabolic-related gene pairs signature analysis identifies ABCA1 expression levels on tumor-associated macrophages as a prognostic biomarker in primary IDHWT glioblastoma. Frontiers in Immunology, 13, 869061.

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Multicolor Flow Cytometry of Mouse Leukocytes

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Mouse peripheral blood leukocytes were stained with antibodies for 30 minutes in the dark, including fluorescently-conjugated antibodies specific to CD45.2 (BD Biosciences; clone-30-F11), CD3 (Biolegend; clone-145–2C11), B220 (Biolegend; clone-RA3–6B2), CD25 (Biolegend; clone-PC61.5), CD8b (Biolegend; clone-YTS156.7.7), CD4 (Biolegend; clone-GK1.5), and NK1.1 (Biolegend; clone-PK136). Cells were then washed with FACS buffer (PBS, 2% FBS, 2mM EDTA) and resuspended in FACS buffer before performing multicolor flow cytometric analysis using a FACS CantoII machine (BD Biosciences). Flow cytometry data were analyzed using FlowJo software version 10 (Tree Star).

Ingle H., Lee S., Ai T., Orvedahl A., Rodgers R., Zhao G., Sullender M., Peterson S.T., Locke M., Liu T.C., Yokoyama C.C., Sharp B., Schultz-Cherry S., Miner J.J, & Baldridge M.T. (2019). Viral complementation of immunodeficiency confers protection against enteric pathogens via IFN-λ. Nature microbiology, 4(7), 1120-1128.

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