After injection of activated BMDCs pulsed with αMHC into WT mice on days 0, 2, and 4, mice were randomly allocated to either the anti-IL-3–treated group or the control IgG-treated group. Neutralizing antibody against IL-3 (100 µg; clone MP2-8F8; Bio X Cell) or control IgG1 (100 µg; clone HRPN; Bio X Cell) was intraperitoneally injected once a day for 7 d.
Clone hrpn
Manufactured by BioXCell
Sourced in United States
The Clone HRPN is a laboratory equipment product. It is designed to perform a core function, but details on its intended use are not provided.
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Lab products found in correlation
Clone rmp1 14, by BioXCell (2 mentions)
Clone 2a3, by BioXCell (2 mentions)
Clone mp5 20f3, by BioXCell (1 mentions)
Clone 5a1, by BioXCell (1 mentions)
Clone afs98, by BioXCell (1 mentions)
Clone mp2 8f8, by BioLegend (1 mentions)
Rat anti mouse il 4 clone 11b11, by BioLegend (1 mentions)
Clone xmg1, by BioXCell (1 mentions)
C57bl 6 mice, by Inotiv (1 mentions)
Anti vegfr2 clone dc101, by BioXCell (1 mentions)
Cxcr2 inhibitor sb225002, by Selleck Chemicals (1 mentions)
Anti il 10 clone jes5 2a5, by BioXCell (1 mentions)
C57bl 6j, by Jackson ImmunoResearch (1 mentions)
10 protocols using clone hrpn
1
Evaluating Anti-IL-3 Therapy in BMDC Immunotherapy
2
Modulating Tumor Growth in Ret Mice
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C57BL/6 mice were subcutaneously (s.c.) injected with 2×104 Ret cells OE IL-6 or not. Tumor size was measured by a caliper and recorded three times per week. On a tumor length or width of 1.5 cm or any other termination criterion, mice were sacrificed and recorded as died. Tumor volume was calculated according to the formula: .25 (link) After 3 weeks, mouse tumors were isolated for FACS analysis.
On the first signs of tumors, RET transgenic mice were separated into four groups containing equal numbers of males and females. One group received isotype control antibodies (clone 2A3, 12.5 mg/kg; and clone HRPN,10 mg/kg; both BioXcell), the second group was injected with anti-PD-1 antibodies (clone RMP1-14, 12.5 mg/kg; BioXcell). Other mice received anti-IL-6 antibodies (clone MP5-20F3, 10 mg/kg; BioXcell) or the combination of anti-PD-1 and anti-IL-6. Antibodies were injected intraperitoneally for 4 weeks, twice per week. Mice with any of the termination criteria were sacrificed and recorded as died. In another set of experiments, mice of the same groups were sacrificed after 4 weeks of therapy, and tumors were isolated for FACS analysis.
On the first signs of tumors, RET transgenic mice were separated into four groups containing equal numbers of males and females. One group received isotype control antibodies (clone 2A3, 12.5 mg/kg; and clone HRPN,10 mg/kg; both BioXcell), the second group was injected with anti-PD-1 antibodies (clone RMP1-14, 12.5 mg/kg; BioXcell). Other mice received anti-IL-6 antibodies (clone MP5-20F3, 10 mg/kg; BioXcell) or the combination of anti-PD-1 and anti-IL-6. Antibodies were injected intraperitoneally for 4 weeks, twice per week. Mice with any of the termination criteria were sacrificed and recorded as died. In another set of experiments, mice of the same groups were sacrificed after 4 weeks of therapy, and tumors were isolated for FACS analysis.
3
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.
4
Lyn Deficiency Cytokine Injection Procedure
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Aged Lyn–/– mice (min. 25 weeks old) were injected intraperitoneally with 100 µg of isotype control (rat IgG1,κ; clone HRPN, BioXcell), 50 µg43 (link) of rat anti-mouse IL-3 (clone MP2-8F8, Biolegend) or 100 µg70 (link) of rat anti-mouse IL-4 (clone 11B11, Biolegend) 72, 48 and 24 h before analysis.
5
Inhibition of IL-3 in Mice
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C57BL/6J, Pf4-Cre-tdTdTomato, or c-mpl−/− mice were injected i.p. once daily for 3 or 5 days with either a neutralizing anti–mouse IL-3 antibody (100 mg, clone MP2-8F8, Bio X Cell) or control IgG1 (100 mg, clone HRPN, Bio X Cell).
6
Immune Cell Depletion and Neutralization Protocols
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Depletion and neutralization experiments were based on previously established protocols in mice. Neutralization of the early IFN-γ burst was done by a single high dose (1 mg) injection of an anti-IFN-γ neutralizing rat-anti mouse monoclonal antibody (clone R4-6A2; BioXCell, USA) or an IgG1 isotype control antibody (clone HRPN; BioXCell, USA) one day prior to infection [48 (link),49 (link)]. In C57Bl/6 mice, NK and NKT cells were depleted using anti-NK1.1 depleting rat anti-mouse monoclonal antibody (clone PK136; BioXCell, USA). One hundred μg of the antibody or an IgG2a isotype control antibody (clone C1.18.4; BioXCell, USA) was given intraperitoneally (i.p.) on days -7, -4, -1, 1, 4 and 7 [50 (link),51 (link)]. For neutralization of CD1d presentation to NKT cells, BALB/c and C57Bl/6 mice were treated i.p. with 75 μg neutralizing anti-mouse CD1d antibody (clone 19G11; BioXCell, USA) or an IgG1 isotype control (clone HRPN; BioXCell, USA) on days -7, -4, -1, 1, 4 and 7 [52 (link)]. For the depletion of neutrophils, BALB/c mice received 75 μg of depleting anti-Ly-6G antibody (clone 1A8) (BioXCell, USA) or isotype IgG2a isotype control antibody (clone 2A3; BioXCell, USA) i.p. on days -4, -1, 1, 4 and 7 [53 (link),54 (link)]. Experiments were performed in duplicate with 3 mice per infection group.
7
Xenograft Tumor Growth Monitoring and Immunotherapy
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All of the animal procedures used were approved by the institutional animal care and use committee at the University of Chicago. Nude mice were purchased from Harlan Sprague-Dawley. C57BL/6 mice were obtained from Envigo. NSG (severely combined immunodeficient (NOD/SCID) interleukin-2 receptor (IL-2R) gamma chain null) mice were obtained from Jackson Laboratory. For xenograft experiments, one million cells were injected subcutaneously into the right flanks of 6-week-old female nude or C57BL/6 mice. Tumor growth was monitored and measured weekly by a caliper, and tumor volume was calculated using the formula, Tumor volume (mm3) = d2 × D/2, where d and D are the shortest and the longest diameters, respectively. For treatment with anti-PD-1 antibody (BioXCell, clone RMP1-14) or isotype control IgG antibody (BioXCell, clone 2A3), B16F10 melanoma cells (5 × 105) were inoculated subcutaneously into C57BL/6 or NSG mice. When the tumors reached a volume of 80–100 mm3, mice were treated with anti-PD-1 or isotype control antibody (200 μg/mouse) by i.p. injection, every other day for three times. For IFNγ blockade treatment, C57BL/6 mice were treated with anti-IFNγ antibody (BioXcell, Clone XMG1.2) or isotype control IgG (BioXcell, Clone HRPN) (250 μg/mouse) every other day after tumor cell inoculation50 (link),51 (link).
8
Depletion of Immune Cell Populations in Tumor Models
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Anti-VEGFR2 (clone DC101, BioXCell, cat# BE0060) or isotype control antibody (clone HRPN, BioXCell, cat# BE0088) was administered i.p. every 3 days starting from day 4 of tumor growth at a dose of 40 mg/kg body weight.
For macrophage depletion, CSF1R inhibitor PLX5622 was administered via rodent chow (1,200 mg PLX5622/kg chow) starting from day 6 of tumor growth. PLX5622 was provided by Plexxikon. Control and PLX5622-containing AIN-76A rodent chow was prepared by Research Diets.
For CD8 þ T-cell depletion, 200 mg CD8-specific antibody (clone YTS169, provided by Polpharma Biologics) was injected i.p. every 2 to 3 days starting 1 day prior to tumor inoculation.
For neutrophil depletion, mice received daily i.p. injections of 200 mg CXCR2 inhibitor (SB225002, Selleckchem, cat# S7651) dissolved in saline with 5% DMSO and 8% Tween-80 starting from the day of tumor implantation. In addition, starting from day 12 of tumor progression, mice received 75 mg Ly6G-specific antibody (clone 1A8, BioXCell, cat# BE0075-1) i.p. every second day, followed by 150 mg anti-rat immunoglobulin (clone MAR 18.5, BioXCell, cat# BE0122) i.p. 24 hours later. The anti-Ly6G treatment was only maintained for 6 days due to the development of anti-rat antibodies in treated mice after 1 week, which limits its efficacy (27) .
For macrophage depletion, CSF1R inhibitor PLX5622 was administered via rodent chow (1,200 mg PLX5622/kg chow) starting from day 6 of tumor growth. PLX5622 was provided by Plexxikon. Control and PLX5622-containing AIN-76A rodent chow was prepared by Research Diets.
For CD8 þ T-cell depletion, 200 mg CD8-specific antibody (clone YTS169, provided by Polpharma Biologics) was injected i.p. every 2 to 3 days starting 1 day prior to tumor inoculation.
For neutrophil depletion, mice received daily i.p. injections of 200 mg CXCR2 inhibitor (SB225002, Selleckchem, cat# S7651) dissolved in saline with 5% DMSO and 8% Tween-80 starting from the day of tumor implantation. In addition, starting from day 12 of tumor progression, mice received 75 mg Ly6G-specific antibody (clone 1A8, BioXCell, cat# BE0075-1) i.p. every second day, followed by 150 mg anti-rat immunoglobulin (clone MAR 18.5, BioXCell, cat# BE0122) i.p. 24 hours later. The anti-Ly6G treatment was only maintained for 6 days due to the development of anti-rat antibodies in treated mice after 1 week, which limits its efficacy (27) .
9
Anti-IL-10 Neutralization in Endotoxemia
10
Immunization of Transgenic Mice Models
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