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Skii 4

Manufactured by BioLegend
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The SKII.4 is a lab equipment product designed for cell sorting applications. It functions as a high-performance cell sorter, enabling the separation and isolation of specific cell populations from complex samples.

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Lab products found in correlation

Facscanto 2 flow cytometer, by BD (1 mentions) Human fcr blocking reagent, by Miltenyi Biotec (1 mentions) Diva software, by BD (1 mentions) Rpa t4, by Thermo Fisher Scientific (1 mentions) Pch101, by Thermo Fisher Scientific (1 mentions) Fcs express, by De Novo Software (1 mentions) Rpa t8, by Thermo Fisher Scientific (1 mentions) Foxp3 staining buffer kit, by Thermo Fisher Scientific (1 mentions) Bw264 56, by Miltenyi Biotec (1 mentions) Eh12.2h7, by BioLegend (1 mentions) Facscalibur cytometer, by BD (1 mentions) Fitc conjugated vimentin, by Abcam (1 mentions) Lsrfortessa, by BD (1 mentions) Mih42, by BioLegend (1 mentions) P1e6 c5, by BioLegend (1 mentions) Pe conjugated streptavidin, by Thermo Fisher Scientific (1 mentions) Human trustain fcx, by BioLegend (1 mentions) Lsrii cytometer, by BD (1 mentions) 4 6 diamidino 2 phenylindole dihydrochloride dapi, by BioLegend (1 mentions) Macsquant x, by Miltenyi Biotec (1 mentions)

4 protocols using skii 4

1

Comprehensive Immune Profiling of T Cells

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Antibodies against human CD3 (BW264/56) and CD45RA (T6D11) were purchased from Miltenyi Biotec. Antibodies against human CD3 (UCHT1), CD25 (BC96), CD127 (A019D5), PD-1 (EH12.2H7), CCR7 (G043H7), and CD155 (SKII.4) were obtained from Biolegend. Fixable viability dye and antibodies against human CD4 (RPA-T4), CD8α (RPA-T8), and Foxp3 (PCH101) were purchased from Thermo Fisher Scientific, and antibody against human CD96v2 (628211) was obtained from R&D Systems. Fluorescently labeled F(ab′)2 against FcγRI (10.1), FcγRIIA/B (AT10), and FcγRIIIA (3G8) were provided by Mark Cragg, University of Southampton. FcγR staining was performed in PBS/1% BSA, without a prior FcγR blocking step. For CFSE proliferation assays and analysis of CD96 and CD155 expression, cells were incubated for 10 minutes at 4°C with 10% heat-inactivated AB serum (MilliporeSigma) prior to surface staining. For TIL phenotyping, cells were incubated with human FcR blocking reagent (Miltenyi Biotec) for 10 minutes at 4°C prior to surface staining. When required, intracellular staining was performed using the Foxp3 staining buffer kit (Thermo Fisher Scientific). Samples were analyzed with a FACSCanto II flow cytometer and DIVA Software (BD Biosciences), FCS Express (De Novo Software), or FlowJo software (version 10).
Rogel A., Ibrahim F.M., Thirdborough S.M., Renart-Depontieu F., Birts C.N., Buchan S.L., Preville X., King E.V, & Al-Shamkhani A. (2022). Fcγ receptor–mediated cross-linking codefines the immunostimulatory activity of anti-human CD96 antibodies. JCI Insight, 7(19), e158444.
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2

Multiparametric Analysis of Cultured Cells

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Cultured cells were harvested and incubated with PerCP Cy5.5-conjugated CD90 (mouse clone Thy1, BioLegend), FITC-conjugated Vimentin (mouse clone RV202, ab8978, Abcam), PE-conjugated PVR (mouse clone SKII.4, BioLegend) or appropriate isotype control at 4°C for 30 min, washed twice, and analyzed using a BD FACS Calibur cytometer. The results were analyzed using Kaluza software.
Inoue T., Adachi K., Kawana K., Taguchi A., Nagamatsu T., Fujimoto A., Tomio K., Yamashita A., Eguchi S., Nishida H., Nakamura H., Sato M., Yoshida M., Arimoto T., Wada-Hiraike O., Oda K., Osuga Y, & Fujii T. (2016). Cancer-associated fibroblast suppresses killing activity of natural killer cells through downregulation of poliovirus receptor (PVR/CD155), a ligand of activating NK receptor. International Journal of Oncology, 49(4), 1297-1304.
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3

Quantitative Flow Cytometry Analysis

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Flow cytometry analysis was performed on an LSRFortessa (BD, USA). Cells were grown in the medium indicated previously, harvested by using Stem Cell Pro Accutase (Invitrogen). Phycoerythrin (PE)-conjugated monoclonal antibodies WM15, IM7 (both Invitrogen), CC2C6, MIH42, MIH2, JS11, SKII.4, P1E6-C5, GoH3, and SHM16 (all Biolegend) directed against CD13, CD44, CD47, CD276/B7-H3, CD274/PD-L1, CD55, CD155, CD49b, CD49f, and EPHA2 were used for detection of osteosarcoma surface markers. PE-conjugated MOPC-21 (Biolegend) were used as isogenic control. A minimum of 30,000 events were recorded. Quantification was performed using BD Quantibrite™ PE-conjugated beads (BD Biosciences) according to the manufacturer’s protocol. Quantitative data is provided (Supplemental Figure 4).
Byrgazov K., Anderson C., Salzer B., Bozsaky E., Larsson R., Gullbo J., Lehner M., Lehmann F., Slipicevic A., Kager L., Fryknäs M, & Taschner-Mandl S. (2020). Targeting aggressive osteosarcoma with a peptidase-enhanced cytotoxic melphalan flufenamide. Therapeutic Advances in Medical Oncology, 12, 1758835920937891.
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4

Multiparametric Flow Cytometry for CAR T-cell Analysis

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Before staining with relevant fluorochrome-conjugated Abs, cells were treated with Human TruStain FcX (Fc receptor blocking solution; BioLegend, San Diego, CA, USA, #422302). Abs against CD4 (APC; OKT4; #317416), CD8 (PerCP-Cy5.5; HIT8a; #300924), TIGIT (PE-Cy7; A15153G; #372713), CD226 (FITC; 11A8; #338303), CD155 (Alexa Fluor 647; SKII.4; #337621), CD111 (PE; R1.302; #340404), HER2 (PE; 24D2; #324405) and mouse IgG1 κ isotype control (PE; MOPC-21; #400113) were from BioLegend, San Diego, CA, USA. Ab against CD96 (PE; NK92.39) was from eBioscience, Thermo Fisher Scientific, Waltham, MA, USA (#12-0969-42) or BioLegend, San Diego, CA, USA (#338405). Cell surface expression of CAR was detected by incubation with recombinant biotinylated protein L (ACROBiosystems, Newark, DE, USA, #RPL-P814R) followed by incubation with PE-conjugated streptavidin (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA, #12-4317-87). To exclude dead cells, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Biolegend, San Diego, CA, USA, #422801) was used. Samples (at least 20,000 events) were acquired on a LSRII cytometer (BD Biosciences) or MACSQuant X (Miltenyi Biotec) and analyzed with FlowJo software (TreeStar, BD Life Sciences, Franklin Lakes, NJ, USA).
Wang C.Q., Choy F.C., Sanny A., Murakami T., Tan A.H, & Lam K.P. (2023). An Inhibitory Role for Human CD96 Endodomain in T Cell Anti-Tumor Responses. Cells, 12(2), 309.
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