2 × 105 plaque-forming units (PFUs) of LCMV-Armstrong were administered intraperitoneally in 8–12 week-old Prf−/− or Prf-Tmem178 double knockout mice. To deplete monocytes/macrophages, 100 μl of clodronate-liposomes were intravenously injected into Prf−/− mice two days prior to the LCMV infection and 48 and 96 h later. Alternatively, 1 mg of the neutralizing anti-CSF1 antibody (Clone 5A1, BioXCell) was administered 2 days prior to infection, and 0.5 mg antibody was administered 48 and 96 h later. Animals were bled via submandibular vein puncture to measure serum cytokines on days 3 and 8 post-infection.
Clone 5a1
Manufactured by BioXCell
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The Clone 5A1 is a laboratory instrument designed for the isolation and purification of specific cell lines. It utilizes advanced cell sorting and separation techniques to generate highly pure cell populations from complex biological samples.
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Lab products found in correlation
5 protocols using clone 5a1
1
LCMV Infection in Perforin Knockout Mice
2
Depletion of Fetal and Postnatal Macrophages
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To deplete YS-derived macrophages pregnant female mice were treated with a single injection of CSF-1R blocking antibody (clone AFS98, Bio X Cell) or rat IgG2a control antibody (clone 2A3, Bio X Cell). Three mg of the antibodies in sterile PBS were administered i.p. to pregnant females at E6.5, as described12 (link),32 (link). Mice were sacrificed at E17.5 or at postnatal age of 2 wk or 5 wk for flow cytometric analyses.
To deplete tissue-resident macrophages after birth, 2 wk old C57Bl/6N mice were cyclically treated with anti-CSF1 antibody and clodronate (Fig.2b ). To that end, three doses of CSF1 neutralizing antibody (Clone 5A1, BioXcell) or control IgG (clone HRPN, BioXcell) were given i.p. (0.5 mg on postnatal day 14, 0.25 mg on postnatal day 18 and 0.25 mg on postnatal day 22). On subsequent days, three doses of clodronate or control liposomes (Liposoma) 50 µl/injection on postnatal days 15, 19 and 23) were administered i.v. The mice were sacrificed 1 or 11 days after the final clodronate treatment. In control experiments using kidney, we saw a full recovery of a known bone marrow-derived CD11b+F4/80Int macrophage population, but not that of a known fetal-derived CD11bIntF4/80Hi macrophage population, verifying the robustness of the model.
To deplete tissue-resident macrophages after birth, 2 wk old C57Bl/6N mice were cyclically treated with anti-CSF1 antibody and clodronate (Fig.
3
Macrophage Depletion in Neonatal Mice
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One-week-old pups were intraperitoneally (i.p.) injected with anti-murine M-CSF antibody (20 μg/g of body weight; clone 5A1, Bio X Cell, West Lebanon, New Hampshire, USA) every third day, receiving 6 injections in total. In parallel, control mice were injected with equal amount of isotype control antibody (clone: HRPN, Bio X Cell).
4
Modulation of Macrophages in Mice
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8–10 weeks old male C57Bl/6 mice were treated by i.p. injection with 1 mg per mouse of neutralizing anti-CSF1 antibody (Clone 5A1, BioXCell, USA) or isotype control (BE0088, BioXCell, USA) and 4 days later with 0.5 mg/mouse antibody given i.p. as described by DeNardo et al [22] (link). At day 7 after initiation of treatment animals were administered radiolabeled HRG for biodistribution analysis. Blood was taken from control animals prior to treatment, at 3 and 7 days after treatment, for endogenous HRG analysis. After 7 days, organs were harvested for immunofluorescent analysis using anti-CD115 (sc-692, Santa Cruz Biotechnology, USA), anti-CD68 (MCA1957AbD Serotec, USA), anti-Ly6G (551459, BD Biosciences, USA), and appropriate fluorescently labeled secondary antibodies (Life Technologies). Immunoblotting to show plasma protein levels was done using anti-Fibrinogen (GAM/Fbg/7S, Nordic Immunology, The Netherlands) and anti-VWF (A0082, Dako, USA) antibodies.
5
Inhibition of CSF-1 Signaling in HLI
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GW2580 (LC Laboratories) was administered once daily by oral gavage at a dose of 80 mg/kg in 0.1% Hydroxipropylmethylcellulose/0.1% Tween-20 as described previously (62 (link)). Treatment started 4 days prior HLI induction. Body weight was measured daily to exclude weight loss. Anti-CSF-1 (aCSF-1) antibody treatment (Clone 5A1, BioXCell) or PBS control was subcutaneously injected in the ischemic limb, at a dose of 50 μg, immediately after surgery or up to 3 days (for details see Figure 3H
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