A0072

Manufactured by Agilent Technologies

Sourced in Canada

The A0072 is a precision laboratory instrument designed for accurate measurements and analysis. It is a core component in various scientific and research applications.

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Lab products found in correlation

Nat105, by Abcam (3 mentions) E1j2j, by Cell Signaling Technology (1 mentions) Pd l1 22c3 pharmdx kit, by Agilent Technologies (1 mentions) Histofine simple stain max po, by Nichirei Biosciences (1 mentions) Bond polymer refine detection kit, by Leica Biosystems (1 mentions) Epr20305, by Abcam (1 mentions) M7103, by Agilent Technologies (1 mentions) Ncl l cd3 565, by Leica camera (1 mentions) Bond 3, by Leica Biosystems (1 mentions) Cd4 4b12, by Agilent Technologies (1 mentions) Emr8 5, by Abcam (1 mentions) Clone 123c3, by Agilent Technologies (1 mentions)

5 protocols using a0072

PD-L1, CD8, and HLA Expression in LUSC

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IHC staining was performed on representative surgical tissue sections from FFPE tissue blocks of LUSC patients using anti–human PD-L1 antibodies: E1J2J, Cell Signaling Technology; 28-8, Abcam; 22C3, Dako; SP142, Spring Bioscience; anti–human CD8 antibodies (C8/144B; Nichirei Bioscience); anti–human PD-1 antibodies (NAT105; Abcam); anti–human β2M antibodies (A0072; Dako); and anti–human HLA class I-A/B/C antibodies (EMR8-5; Hokudo). The detection of immunostaining was performed using Histofine Simple Stain MAX-PO (Nichirei Bioscience) or BOND polymer Refine Detection kit (Leica Biosystems). Some of the sections were stained with the PD-L1 22C3 pharmDx kit (Dako). PD-L1 positivity was defined as membranous or cytoplasmic staining in at least 1% of tumor cells. The staining results of β2M and HLA class I-A/B/C of tumor cells were categorized as negative (0%), focally positive (<50%), or positive (≥50%).

Sagawa R., Sakata S., Gong B., Seto Y., Takemoto A., Takagi S., Ninomiya H., Yanagitani N., Nakao M., Mun M., Uchibori K., Nishio M., Miyazaki Y., Shiraishi Y., Ogawa S., Kataoka K., Fujita N., Takeuchi K, & Katayama R. (2022). Soluble PD-L1 works as a decoy in lung cancer immunotherapy via alternative polyadenylation. JCI Insight, 7(1), e153323.

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Multiplex IHC for Immune Profiling

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IHC staining was performed on representative tissue sections from formalin-fixed and paraffin-embedded tissue blocks using anti-human CD8 antibodies (C8/144B; Nichirei Biosciences), anti-human PD-1 antibodies (NAT105; Abcam), anti-human HLA class I-A, B, and C antibodies (EMR8-5; HoKudo), anti-human B2M antibodies (A0072; Dako), anti-mouse CD8 antibodies (EPR20305; Abcam), anti-mouse PD-1 antibodies (D7D5W; Cell Signaling Technology), and anti-mouse granzyme B antibodies (D6E9W; Cell Signaling Technology).

Gong B., Kiyotani K., Sakata S., Nagano S., Kumehara S., Baba S., Besse B., Yanagitani N., Friboulet L., Nishio M., Takeuchi K., Kawamoto H., Fujita N, & Katayama R. (2019). Secreted PD-L1 variants mediate resistance to PD-L1 blockade therapy in non–small cell lung cancer. The Journal of Experimental Medicine, 216(4), 982-1000.

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Soluble HLA-A*02:01 Purification and Peptide Isolation

Cited 1 time

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The soluble HLA-A*02:01 was transfected into different cell lines (HeLa, A375, SKOV3, A2780 and OV90). Transfected cells were single cell sorted (subcloned) to identify a high expressing clone. Soluble HLA constructs are truncated at the 3′end of exon 4, deleting the transmembrane and cytoplasmic domains, and include a C-terminal VLDLr epitope purification tag (SVVSTDDDLA) that is recognized by the anti-VLDLr mAb (ATCC CRL-2197). This antibody was used both for purification of soluble HLA from cells supernatant and for quantification of sHLA production as the capture antibody in a sandwich ELISA, with an antibody directed against β2-microglobulin (Dako A0072) as the detector antibody. After transfection, cells were grown at high density in hollow-fiber bioreactors (AcuSyst-Maximizer, C3 Cell Culture Company) and sHLA/peptide complexes were purified from supernatants by affinity chromatography with the anti-VLDLr antibody. Eluate fractions containing sHLA/peptide complexes were pooled, brought to a final acetic acid concentration of 10%, and heated to 78°C in a water bath to denature HLA. Peptides were purified and isolated from alpha chain and B2m using an Ultracel 3 kDa cutoff cellulose membrane (EMD Millipore, PLBC06210) and lyophilized (Carreno et al., 2015 (link); Patterson et al., 2016 (link)).

Marty R., Kaabinejadian S., Rossell D., Slifker M.J., van de Haar J., Engin H.B., de Prisco N., Ideker T., Hildebrand W.H., Font-Burgada J, & Carter H. (2017). MHC-I Genotype Restricts the Oncogenic Mutational Landscape. Cell, 171(6), 1272-1283.e15.

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Comprehensive IHC Profiling of CRCs

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IHC staining for B2M using a polyclonal antibody with concentration of 1:6,000 (catalog #A0072; Dako,
Santa Clara, CA), MHC class I using a monoclonal antibody with concentration of 1:200 (catalog #14–9958; E-Bioscience,
Carlsbad, CA), CD3 using a monoclonal antibody with concentration of 1:200 (catalog #NCL-L-CD3–565; Leica, Lincolnshire,
IL), CD8 using a monoclonal antibody with concentration of 1:100 (catalog #M7103; Dako), and PD-L1 using a monoclonal antibody
with concentration of 1:100 (catalog #13684; Cell Signaling, Danvers, MA) was performed on all CRCs with B2Mmutations with available tissue as well as a set of 26 randomly selected wild-type (WT) CRCs with in-house resection specimens and
MSK-IMPACT testing (performed between January 1, 2014, and October 31, 2017) that were matched to the B2M-mutant
group for prevalence of MSI status. Levels of CD3⁺ and CD8⁺ TILs were assessed via the average of five
counted fields per patient case at ×400 original magnification on light microscopy. IHC-positive cells were counted up to a
maximum of 150 cells, because counts > 150 per high-powered field (HPF) tended to have clustering, which led to difficulty
establishing accurate counts. B2M and MHC class I expression were each recorded as retained or lost for each
patient case. Complete loss of B2M on IHC (0% of tumor cells with B2M expression) was
interpreted as loss of B2M expression.

Middha S., Yaeger R., Shia J., Stadler Z.K., King S., Guercio S., Paroder V., Bates D.D., Rana S., Diaz LA J.r., Saltz L., Segal N., Ladanyi M., Zehir A, & Hechtman J.F. (2019). Majority of B2M-Mutant and -Deficient Colorectal Carcinomas Achieve Clinical Benefit From Immune Checkpoint Inhibitor Therapy and Are Microsatellite Instability-High. JCO Precision Oncology, 3, PO.18.00321.

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Comprehensive Immune Profiling of Melanoma

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Dual IHC for MHC class I (HLA-A, HLA-B, and HLA-C, clone EMR8-5, 1:6000; Abcam) or MHC class II (HLA-DP, HLA-DQ, HLA-DR, clone CR3/43, 1:750; Dako) with the melanoma marker SOX10 (EP268, 1:1500; Cell Marque) was performed using an automated staining system (Bond-III; Leica Biosystems), as previously described (42) .
IHC for CD3 (LN10; Leica), CD4 (4B12; Dako), CD8 (C8/144B; Dako), PD-1 (NAT105; Abcam), CD56 (clone 123C3; Dako), TCR (clone H-41; Santa Cruz Biotechnology), and 2M (A0072, 1:6000; Dako) was performed either manually (CD3, CD4, CD8, and PD-1) or on Bond RX (2M, CD56, and TCR) per standard protocols. IHC for PD-L1 (28-8; Dako) was performed as part of an investigational use-only kit (PD-L1 IHC pharmDx) on Dako Link 48 (2) .

Rodig S.J., Gusenleitner D., Jackson D.G., Gjini E., Giobbie-Hurder A., Jin C., Chang H., Lovitch S.B., Horak C., Weber J.S., Weirather J.L., Wolchok J.D., Postow M.A., Pavlick A.C., Chesney J, & Hodi F.S. (2018). MHC proteins confer differential sensitivity to CTLA-4 and PD-1 blockade in untreated metastatic melanoma. Science translational medicine, 10(450).

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