Hit3a

Manufactured by BioLegend

Sourced in United States

The HIT3a is a laboratory equipment product offered by BioLegend. It is a high-quality instrument designed for specific laboratory applications. The core function of the HIT3a is to perform a particular set of tasks within the research or analytical workflow. No further details can be provided while maintaining an unbiased and factual approach.

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38 protocols using hit3a

Transfection and Activation of Human Cell Lines

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HEK 293T cells were cultured in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and transfected using polyethylenimine (PEI) (Polysciences) according to the manufacturer's instructions. Human‐derived Jurkat T cells were maintained in RPMI 1640 medium containing 10% FBS. Transfection of Jurkat cells with plasmid DNA was performed by electroporation on a Gene Pulser X cell apparatus (Bio‐Rad Laboratories, USA). Jurkat T cells were activated using PMA/ionomycin or soluble antibodies against CD3 (1 μg/ml, Hit3a, BioLegend, USA) and CD28 (1 μg/ml, CD28.2, BioLegend, USA). Cell lines were obtained from the laboratory of Drew Pardoll (Johns Hopkins) and were not verified experimentally.

Yang J., Wei P., Barbi J., Huang Q., Yang E., Bai Y., Nie J., Gao Y., Tao J., Lu Y., Xie C., Hou X., Ren J., Wu X., Meng J., Zhang Y., Fu J., Kou W., Gao Y., Chen Z., Liang R., Tsun A., Li D., Guo W., Zhang S., Zheng S., Niu J., Galardy P., Tong X., Shi G., Li H., Pan F, & Li B. (2020). The deubiquitinase USP44 promotes Treg function during inflammation by preventing FOXP3 degradation. EMBO Reports, 21(9), e50308.

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Cell Surface Immunophenotyping Protocol

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For cell-surface labeling, 1 × 10⁶ cells were blocked with Fc-block reagent (BD Biosciences). Then the following antibodies were added and incubated for 30 min, including anti-CD3 (BioLegend, HIT3a), anti-CD14 (BioLegend, 63D3), anti-HLA-DR (BioLegend, L243), and anti-CD45 (BioLegend, 2D1). After incubation, the samples were washed and reconstituted in phosphate-buffered saline for flow cytometric analysis on a FACSCanto II flow cytometer.

Xu G., Qi F., Li H., Yang Q., Wang H., Wang X., Liu X., Zhao J., Liao X., Liu Y., Liu L., Zhang S, & Zhang Z. (2020). The differential immune responses to COVID-19 in peripheral and lung revealed by single-cell RNA sequencing. Cell Discovery, 6, 73.

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Isolation and Activation of Human Naive CD8+ T Cells

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Peripheral blood mononuclear cells (PBMCs) from healthy human donors were obtained from the CFAR Virology Core at UCLA without personal identifying information. To minimize a potential induction of T-cell stimulation by events such as crosslinking of cell-surface molecules, T_N-cell population was negatively selected in one step using an EasySep™ Human Naive CD8+ T Cell Enrichment Kit (StemCell Technologies, Inc., Vancouver, Canada), which consistently provides >95% purity of CD8+ T_N cells (Supplementary Figure 1). Cells were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 1% GlutaMAX supplement and Antibiotic-Antimycotic (Life Technologies, Grand Island, NY), 20% FCS (SH30070.03E; GE Healthcare Life Sciences, South Logan, UT), and 0.1 mM 2-mercaptoethanol (Sigma-Aldrich St. Louis, MO) (T-cell medium) as reported previously (27 (link)). Prior to lentiviral vector transduction, CD8+ T_N cells were incubated with various concentrations of anti-CD3 antibody (Hit3a; BioLegend, San Diego, CA) with or without 2 µg/mL of anti-CD28 antibodies (CD28.2; BioLegend) for 2 days in T-cell medium supplemented with 5 ng/mL IL-7 and IL-15 (R&D systems, Minneapolis, MN) as summarized in Figure 1A. All cells were incubated at 37°C in 5% CO₂.

Kranz E., Kuhlmann C.J., Chan J., Kim P.Y., Chen I.S, & Kamata M. (2022). Efficient derivation of chimeric-antigen receptor-modified TSCM cells. Frontiers in Immunology, 13, 877682.

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Comprehensive Flow Cytometry for Tumor Immune Profiling

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Single cell suspensions of tumor and tumor-adjacent tissue were stained
with surface antibodies for CD19, CD20, CD3, CD8, CD4, CD56 and CD11c (clones
HIB19, 2H7, HIT3a, RPA-T8, RPA-T4, HCD56, and 3.9, respectively, Biolegend) and
an intracellular stain for the transcription factor FoxP3 (clone PCH101,
Ebioscence) for Figure 1 and for CD19, CD20, CD21, CD69, CD27 and HLA-DR (clones HIB19, 2H7, HB5, FN50, O323, and L243,
respectively, Biolegend and Ebioscience) for Figs. 2 and 3. Positive and negative
controls for staining were PBLs from patients or control donors, lymphocytes
from control donor spleens, lymphocytes from control (disease-free) lungs,
lymphocytes from NSCLC pleural metastases, and PBL from untreated,
HIV⁺ patients. Flow cytometry was conducted using an LSR
II (BD Biosciences) and data were analyzed using FlowJo software (Tree Star,
Inc.). For Fig. 2B, total lymphocyte infiltration was calculated using the following equation: [Total cell
number from tissue X % lymphocyte gate X % lymphocyte
population, i.e. CD19⁺]/ [mass of tissue in
grams].

Bruno T.C., Ebner P.J., Moore B.L., Squalls O.G., Waugh K.A., Eruslanov E.B., Singhal S., Mitchell J.D., Franklin W.A., Merrick D.T., McCarter M.D., Palmer B.E., Kern J.A, & Slansky J.E. (2017). Antigen-presenting intratumoral B cells affect CD4+ TIL phenotypes in non-small cell lung cancer patients. Cancer immunology research, 5(10), 898-907.

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Comprehensive immune cell analysis

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The following antibodies were used: anti-human CD3 (HIT3a, Biolegend), anti-human CD8 (SK1, Biolegend), anti-human NKp30 (P30-15, Biolegend), anti-human HLA-A2 (BB7.2, Biolegend), anti-human HER2 (24D2, Biolegend), anti-mouse TCR-β chain (H57-597, Biolegend), anti-human CD107a (H4A3, Biolegend), anti-human IFN-γ (B27, Biolegend). Corresponding isotype controls mIgG1 (MOPC-21, Biolegend), mIgG2b (MG2b-57, Biolegend) and Armenian Hamster IgG (HTK888, Biolegend) were used. Anti-B7H6 clone 1.18 and corresponding isotype control were from in-house production.^{9 (link)} For CD8⁺ T cell activation, anti-human functional-grade purified anti-CD3 (UCHT1, Biolegend) and anti-CD28 (37.51, Biolegend) were used.

Correia M.P., Stojanovic A., Wels W.S, & Cerwenka A. (2021). Innate-like NKp30+CD8+ T cells armed with TCR/CAR target tumor heterogeneity. Oncoimmunology, 10(1), 1973783.

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Monitoring Glycolytic Shift in T-Cell Activation

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To monitor the glycolytic switch upon activation, CD4⁺ and CD8⁺ T-cells were resuspended in serum-free XF Assay media supplemented with 11.1 mM glucose and 2 mM l-glutamine (Sigma). ECAR and OCR were measured simultaneously throughout the experiment, i.e., 1 h before activation and 4 h after. T-cells were activated via the multi-injection port with anti-CD3 (0.2 µg/ml; HIT3a, BioLegend) and CD28 (20 µg/ml; CD28.2, BioLegend). A final injection of 2-DG (100 mM) was used to immediately arrest glycolysis. Isotype controls, mIgG2a κ (0.2 µg/ml; MOPC-173, BioLegend) and mIgG1 κ (20 µg/ml; MOPC-21; BioLegend) were used. The OCR/ECAR ratio was calculated by dividing the eight corresponding OCR and ECAR measurements pre- (dotted boxes) or post- (dashed boxes) antibody injection. Fold ECAR change was calculated by dividing the single point post antibody injection by the single point pre antibody injection. Peptide stimulation relied on the cross presentation of specific peptides by corresponding T-cell clones.

Jones N., Cronin J.G., Dolton G., Panetti S., Schauenburg A.J., Galloway S.A., Sewell A.K., Cole D.K., Thornton C.A, & Francis N.J. (2017). Metabolic Adaptation of Human CD4+ and CD8+ T-Cells to T-Cell Receptor-Mediated Stimulation. Frontiers in Immunology, 8, 1516.

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Comprehensive Antibody Validation Protocol

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Antibodies used in this work included anti‐CD3e (HIT3a, BioLegend), anti‐CD28 (CD28.2, BD Biosciences), anti‐P150‐Glued (1/p150Glued (RUO), AB_397846, BD Biosciences), anti‐CCT1 (91a, AB_2920800, Abcam), anti‐CCT2 (5B5C4, AB_2920801, Merck Millipore), anti‐CCT4 (EPR8495(B), AB_11144306, Abcam), anti‐CCT5 (EPR7562, AB_11154964, Abcam), anti‐CD63 (NKI/C3, AB_2920802, Abcam), anti‐CD81 (5A6, AB_627192, Santa Cruz), anti‐CD63 (Tea3/18, propietary), anti‐Calnexin (AB_2069009, Abcam), anti‐EEA1 (N‐19, AB_2096822, Santa Cruz), anti‐TSG101 (4A10, AB_306450, Abcam), anti‐ADFP (EPR3713, AB_10863476, Abcam), anti‐Pex14 (AB_2736340, Invitrogen), anti‐Alpha‐Tubulin (DM1A, AB_477593, Sigma Aldrich), anti‐Alpha‐Tubulin FITC‐conjugated (DM1A, AB_476968, Sigma Aldrich), anti‐K40 alpha‐tubulin (6‐11B‐1, AB_609894, Sigma Aldrich), anti‐Kinesin Heavy Chain (H1, AB_94283, Merck Millipore), anti‐Kinesin Light Chain (L1, AB_94286, Merck Millipore), anti‐Beta‐Actin (AC‐15, AB_476692, Sigma Aldrich), anti‐TFAM (EPR12286, AB_2920803, Abcam), BODIPY™ 493/503 Neutral lipids (Invitrogen), BODIPY™ c11 581/591 Peroxized lipids (Invitrogen), LysoTracker™ Red DND‐99 (Invitrogen), MitoTracker™ Orange CMTMRos (Invitrogen), MitoTracker™ Green (Invitrogen), Nonyl‐acridine‐orange (Invitrogen), CellTracker™ Blue CMAC Dye (Invitrogen), and Phalloidin Alexa Fluor™ 647 (Invitrogen).

Rojas‐Gómez A., Dosil S.G., Chichón F.J., Fernández‐Gallego N., Ferrarini A., Calvo E., Calzada‐Fraile D., Requena S., Otón J., Serrano A., Tarifa R., Arroyo M., Sorrentino A., Pereiro E., Vázquez J., Valpuesta J.M., Sánchez‐Madrid F, & Martín‐Cófreces N.B. (2023). Chaperonin CCT controls extracellular vesicle production and cell metabolism through kinesin dynamics. Journal of Extracellular Vesicles, 12(6), 12333.

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Isolation and Characterization of Memory T Cells

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FACS was performed to isolate α4⁺ memory T cells for subsequent scRNASeq, with α4⁺ memory T cells being defined as CD3⁺CD45RA⁻α4⁺ viable single lymphocytes. To that end, the following fluorescently-labeled antibodies were used in combination with fixable viability dye eFluor780 (eBioscience (1:1000)): CD3 (APC, HIT3a, Biolegend (1:100)), α4 (PE/Cy7, 9F10, Biolegend (1:100)), CD45RA (VioGreen, REA1047, Miltenyi Biotec (1:100)).
To isolate β7^+/− CD4 (CD3⁺TCRVα7.2⁻CD4⁺CD8a⁻CD45RA⁻ β7^+/− single lymphocytes) and CD8 (CD3⁺TCRVα7.2⁻CD4⁻CD8a⁺CD45RA⁻ β7^+/− single lymphocytes) memory T cells for functional investigations and CD103-inducibility analysis, we used fluorescently-labeled antibodies against the following epitopes: CD3 (PE/Cy7, SK7, Biolegend (1:1000)), TCRVα7.2 (BV421, OF5A12, BD Biosciences (1:500)), CD4 (APC-Vio770, VIT4, Miltenyi Biotec (1:2000)), CD8a (FITC, RPA-T8, Biolegend (1:2000)), CD45RA (VioGreen, REA1047, Miltenyi Biotec (1:1000)), β7 (PE, FIB27, Biolegend (1:1000)).
FACS Aria II SORP (BD Biosciences) instruments were used for FACS.
MACS enrichment for CD4 and CD8 T cells from PBMCs was performed according to manufacturer’s instructions using the CD4 T Cell Isolation Kit, human and CD8 T Cell Isolation Kit, human, respectively (both Miltenyi Biotec).

Wiendl M., Dedden M., Liu L.J., Schweda A., Paap E.M., Ullrich K.A., Hartmann L., Wieser L., Vitali F., Atreya I., Müller T.M., Günther C., Atreya R., Neurath M.F, & Zundler S. (2024). Etrolizumab-s fails to control E-Cadherin-dependent co-stimulation of highly activated cytotoxic T cells. Nature Communications, 15, 1043.

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Multicolor Immunofluorescence Staining of PBMCs, HT-29, and CAR T Cells

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Human PBMCs were stained with anti-human CD3 (HIT3a, BioLegend), allophycocyanin–anti-human CD64 (10.1, BioLegend), and Alexa Fluor 647 anti-human CD16 (3G8, BioLegend). HT-29 were stained with Alexa Fluor 488 anti-human CD340 (erbB2/HER-2, 24D2, BioLegend), CAR T cells were stained by Human HER2/ErbB2 Protein (ECD) Biotinylated (Sino Biological, #10004-HCCH-B) and Alexa Fluor 594 Streptavidin (BioLegend, #405240). Images were collected using a Zeiss LSM800 confocal laser scanning microscope using Objective Plan-Apochromat 20x/0.8 DIC M27 and solid-state laser lines, and analyzed using ZEN software (Carl Zeiss Microscopy, version ZEN 2.6 blue).

Rasoulouniriana D., Santana-Magal N., Gutwillig A., Farhat-Younis L., Tal L., Amar S., Milyavsky M., Muddineni S.S., Solomon N., Shpilt H., Dotan S., Pilpel N., Waskow C., Feinmesser M., Rider P, & Carmi Y. (2023). T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors. Cancer Immunology Research, 11(6), 792-809.

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Multiparameter FACS Staining for Immune Profiling

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For FACS staining, we used the following antibodies to target: CD16 (FITC; eBioCB16), CD14 (PE-Cy7; 61D3), CD15 (APC; MMA), IL-4 (APC; 8D4-8), and IFN-γ (4S.B3)—all purchased from eBioscience (San Diego, CA, USA)—and CD11b (FITC; ICRF44), CD56 (PE; HCD56), CD33 (PE; WM53), IL-10 (PE; JES3-9D7) CD45 (APC; HI30), CD163 (APC; GH1/61), CD3 (PE-Cy7; HIT3a), CD4 (FITC; OKT4), and IL-17A (PE; BL168)—all purchased from BioLegend (San Diego, CA, USA).

Kasper M., Walscheid K., Laffer B., Bauer D., Busch M., Wildschütz L., Wang B., Loser K., Vogl T., Grajewski R.S., Langmann T, & Heiligenhaus A. (2018). The Phenotype of Monocytes in Anterior Uveitis Depends on the HLA-B27 Status. Frontiers in Immunology, 9, 1773.

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