αpd l1 clone 10f 9g2, by BioXCell - Product details

1

Neuro2a Cell Line Culture and Checkpoint Inhibitors

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The murine neuroblastoma cell line Neuro2a is derived from an aggressive and metastatic sub-clone of the C1300 neuroblastoma cell line that was cultured from a spontaneous tumor in the spinal cord of A/J mice (ATCC, Manassas, VA). Neuro2a cells were maintained in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA) and 10% fetal bovine serum (Gemini Bioproducts, Sacramento, CA). Cells were grown at 37°C under 5% CO₂. Anti-mouse checkpoint inhibitors αCTLA-4 (clone 9D9) and αPD-L1 (clone 10F.9G2) were obtained from commercially available source (BioXCell, West Lebanon, NH).

Eranki A., Srinivasan P., Ries M., Kim A., Lazarski C.A., Rossi C.T., Khokhlova T.D., Wilson E., Knoblach S.M., Sharma K.V., Wood B.J., Moonen C.T., Sandler A.D, & Kim P.C. (2019). High Intensity Focused Ultrasound (HIFU) Triggers Immune Sensitization of Refractory Murine Neuroblastoma to Checkpoint Inhibitor Therapy. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(5), 1152-1161.

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2

CD8 T Cell Activation Assay

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CD8 T cells were purified from splenocytes of vaccinated mice using CD8 positive selection kits (Miltenyi Biotec). For IFNγ EliSpot assays, effector cells were incubated at different numbers (adjusted to the number of tetramer+ T cells), together with 1 × 10⁵ stimulator cells (B16F10, B16F10 pulsed with minimal gp33-41 peptide or B16F10gp33-41 cells). In some experiments, B16F10gp33-41 cells were pre-treated with IFNγ (50 ng/ml) for 48 h. For in vitro PD-L1 blockade, 10 μg/ml of αPD-L1 (clone 10F.9G2, BioXcell) was used. ImmunoSpot System (Cellular Technology Ltd, Cleveland, OH) was used to develop and count the cytokine-positive spots. For ELISA, purified CD8 T cells were incubated with different concentrations of minimal gp33-41 or gp34-41 peptides. Two days later, the supernatants were collected and IFNγ production was quantified using an ELISA kit (eBioscience) in accordance with the manufacturer’s protocol.

Sultan H., Trillo-Tinoco J., Rodriguez P, & Celis E. (2017). Effective antitumor peptide vaccines can induce severe autoimmune pathology. Oncotarget, 8(41), 70317-70331.

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3

Tumor Immunotherapy Treatment Regimen

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Two days following tumor detection, mice were enrolled into one of four treatment groups. Control Rat IgG (BioXCell) and αPDL1 (clone 10 F.9G2, BioXCell) antibodies were given intra peritoneally (100 μg/mouse) every other day, αCTLA4 antibody (clone UC10–4 F10–11, BioXCell) was given i.p. (100 μg/mouse) every three days twice. For the long-term treatment, mice were treated for two weeks, and for the mass cytometry experiment, mice were treated for one week. For the macrophage-depletion experiments, anti-CSF1R antibody (#BE0213, clone AFS98, BioCXell) was administered (400 μg/mouse) thrice a week for two weeks (24 (link),25 (link)).

Dhanasekaran R., Hansen A.S., Park J., Lemaitre L., Lai I., Adeniji N., Kuruvilla S., Suresh A., Zhang J., Swamy V, & Felsher D.W. (2023). MYC Overexpression Drives Immune Evasion in Hepatocellular Carcinoma that is Reversible Through Restoration of Pro-Inflammatory Macrophages. Cancer research, 83(4), 626-640.

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Antibody-Mediated Immune Modulation in Mice

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Mice were given intraperitoneal injections of 200 μg of each respective antibody (as outlined in each specific experiment) diluted to a total of 100 μL in sterile PBS using a 27-guage needle. Checkpoint blockade inhibitor antibodies were purchased from BioXCell (West Lebanon, NH, USA); αCTLA-4 clone 9H10 (BE0131), αPD-1 clone 29F.1A12 (BE0273), αPD-L1 clone 10F.9G2 (BE0101), or Isotype clone 2A3 (BE0089). CD8 depletion was performed with the rat anti-mouse αCD8 clone 2.43 and isotype clone 2A3. NK depletion was performed with αNK1.1 clone PK136 (BE0036) and isotype clone C1.18.4 (BE0085), both from BioXCell.

Gilley R.P, & Dube P.H. (2020). Checkpoint blockade inhibitors enhances the effectiveness of a Listeria monocytogenes-based melanoma vaccine. Oncotarget, 11(7), 740-754.

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5

Anti-PD-L1 Therapy for Disease Modulation

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αPD-L1 (clone 10F.9G2; BioXcell) treatment (200ug/mouse) was administered i.p. every 3 days from 22dpi-35dpi for a total of 5 treatments (2 (link)); PBS was used as a vehicle control.

Wu J.E., Manne S., Ngiow S.F., Baxter A.E., Huang H., Freilich E., Clark M.L., Lee J.H., Chen Z., Khan O., Staupe R.P., Huang Y.J., Shi J., Giles J.R, & Wherry E.J. (2023). In Vitro Modeling of CD8 T Cell Exhaustion Enables CRISPR Screening to Reveal a Role for BHLHE40. bioRxiv.

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Chronic LCMV and Tumor Immunotherapy Studies

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For chronic LCMV studies, female C57BL/6 mice (5–6 weeks old) were infected with clone 13 as described above. Mice were injected i.p. with either 200 μg αPD-L1 antibody (clone 10F.9G.2, Bio X Cell) or control PBS every 3 days starting at d22 for 5 total treatments as previously described (Barber et al., 2006 (link)). Spleens were harvested on d35 post-infection and splenocytes were stained and analyzed by flow cytometry and imaging flow cytometry.
For mouse tumor studies, female BALB/C mice (5–6 weeks old) were purchased from the National Cancer Institute. CT26 cells were maintained in RPMI1640 supplemented with 10% FCS, 1% Glutamax, and 1% penicillin/ streptomycin and maintained at 5% CO₂. For primary tumor experiments, 2×10⁵ CT26 cells/100 μL were injected subcutaneously into mice. Cohorts of tumor-bearing mice were then injected with phosphate buffered saline (PBS) or 200 μg of αPD-L1 (clone 10F.9G2, Bio X Cell) on d10, d13, d16, and d19. Established CT26 tumors were excised from mice on d20 post-tumor injection and processed for flow cytometry analysis as previously described (Knight et al., 2013 (link)). Briefly, tumors were digested with 1 mg/ml collagenase D and 0.02 mg/ml DNase I at 37°C. Digested tumors were passed through a cell strainer to prepare a single cell suspension. Cells were then stained for ImageStream analysis as described.

McLane L.M., Ngiow S.F., Chen Z., Attanasio J., Manne S., Ruthel G., Wu J.E., Staupe R.P., Xu W., Amaravadi R.K., Xu X., Karakousis G.C., Mitchell T.C., Schuchter L.M., Huang A.C., Freedman B.D., Betts M.R, & Wherry E.J. (2021). Role of nuclear localization in the regulation and function of T-bet and Eomes in exhausted CD8 T cells. Cell reports, 35(6), 109120.

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Chronic LCMV and Tumor Immunotherapy Studies

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For chronic LCMV studies, female C57BL/6 mice (5–6 weeks old) were infected with clone 13 as described above. Mice were injected i.p. with either 200 μg αPD-L1 antibody (clone 10F.9G.2, Bio X Cell) or control PBS every 3 days starting at d22 for 5 total treatments as previously described (Barber et al., 2006 (link)). Spleens were harvested on d35 post-infection and splenocytes were stained and analyzed by flow cytometry and imaging flow cytometry.
For mouse tumor studies, female BALB/C mice (5–6 weeks old) were purchased from the National Cancer Institute. CT26 cells were maintained in RPMI1640 supplemented with 10% FCS, 1% Glutamax, and 1% penicillin/ streptomycin and maintained at 5% CO₂. For primary tumor experiments, 2×10⁵ CT26 cells/100 μL were injected subcutaneously into mice. Cohorts of tumor-bearing mice were then injected with phosphate buffered saline (PBS) or 200 μg of αPD-L1 (clone 10F.9G2, Bio X Cell) on d10, d13, d16, and d19. Established CT26 tumors were excised from mice on d20 post-tumor injection and processed for flow cytometry analysis as previously described (Knight et al., 2013 (link)). Briefly, tumors were digested with 1 mg/ml collagenase D and 0.02 mg/ml DNase I at 37°C. Digested tumors were passed through a cell strainer to prepare a single cell suspension. Cells were then stained for ImageStream analysis as described.

McLane L.M., Ngiow S.F., Chen Z., Attanasio J., Manne S., Ruthel G., Wu J.E., Staupe R.P., Xu W., Amaravadi R.K., Xu X., Karakousis G.C., Mitchell T.C., Schuchter L.M., Huang A.C., Freedman B.D., Betts M.R, & Wherry E.J. (2021). Role of nuclear localization in the regulation and function of T-bet and Eomes in exhausted CD8 T cells. Cell reports, 35(6), 109120.

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In vivo CD4/CD8 Depletion and PD-L1 Blockade

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For CD4 and CD8 depletion, mice were injected intraperitoneally with 300 µg of anti-CD4 (clone GK1.5, BioXCell) or 300 µg of anti-CD8α (clone 2.43, BioXCell) 3 days before tumor challenge or the day before intravesical treatment. Repeated doses were administered to achieve continuous depletion if needed. For antibody-based treatment, 200 µg of αPD-L1 (clone 10F.9G2, BioXCell) was injected intraperitoneally two times a week.

Moreo E., Uranga S., Picó A., Gómez A.B., Nardelli-Haefliger D., del Fresno C., Murillo I., Puentes E., Rodríguez E., Vales-Gómez M., Pardo J., Sancho D., Martín C, & Aguilo N. (2022). Novel intravesical bacterial immunotherapy induces rejection of BCG-unresponsive established bladder tumors. Journal for Immunotherapy of Cancer, 10(7), e004325.

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9

Lipid Nanoparticle Formulation for Camptothecin Delivery

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(S)-(+)-camptothecin (CPT, 98%) was purchased from BLDpharm (Shanghai, China). Sphingomyelin (egg, 99%), DSPE-PEG_2K (99%), DSPE-Cy5.5 (99%), and cholesterol (ovine, 99%) were purchased from Avanti (Alabama, USA). Dextrose (99%), chlorpromazine (98%), genistein (98%), methyl-β-cyclodextrin (98%), wortmannin (98%), cytochalasin D (98%) and NaN₃ (98%) were purchased from Fisher Scientific (USA). Onivyde^® (Ispsen Biopharmaceuticals, Inc) was acquired from Pharmacy Department, Banner-University Medical Center Tucson. Matrigel (#254248) was purchased from Corning, Discovery Labware. α-PD-L1 (clone 10 F.9G2) and α-IFN-γ (clone R4-6A2) were purchased from BioXCell.

Wang Z., Cordova L.E., Chalasani P, & Lu J. (2022). Camptothesome Potentiates PD-L1 Immune Checkpoint Blockade for Improved Metastatic Triple Negative Breast Cancer Immunochemotherapy. Molecular pharmaceutics, 19(12), 4665-4674.

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αpd l1 clone 10f 9g2

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9 protocols using αpd l1 clone 10f 9g2

Neuro2a Cell Line Culture and Checkpoint Inhibitors

CD8 T Cell Activation Assay

Tumor Immunotherapy Treatment Regimen

Antibody-Mediated Immune Modulation in Mice

Anti-PD-L1 Therapy for Disease Modulation

Chronic LCMV and Tumor Immunotherapy Studies

Chronic LCMV and Tumor Immunotherapy Studies

In vivo CD4/CD8 Depletion and PD-L1 Blockade

Lipid Nanoparticle Formulation for Camptothecin Delivery

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