Analysis of tetramer+ cells was performed as previously described with minor modifications9 (link),36 (link)-38 (link). In brief, cells were blocked with CD16/32 (93) and stained with PE-, APC-, or PE- and APC-conjugated tetramers specific against SFB (SFB3340 200-210, NIH tetramer core facility) at room temperature for 20 min and at 4 °C for 40 min in the dark. Next, cells were incubated with anti-PE or anti-APC nanobeads (BioLegend) for 15 min at 4 °C and the PE+ or APC+ magnetically labelled fraction was retained using a MojoSort Magnet (BioLegend). We then proceeded to surface staining with antibodies as in Extended Data Fig. 1a . Using the same methodology we tested PE-conjugated tetramers specific against 2W1S, Helicobacter hepaticus and human peptides (Ealpha 52-68 variant, HH1713 172-186, and class II-associated invariant chain peptide, NIH tetramer core facility).
Mojosort magnet
Manufactured by BioLegend
Sourced in United States
The MojoSort™ Magnet is a laboratory instrument designed to facilitate magnetic cell separation. It provides a magnetic field to enable efficient isolation and purification of target cells from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd137 rabbit mab, by Cell Signaling Technology (5 mentions)
Mojosort human cd8 nanobeads, by BioLegend (5 mentions)
Anti cd137 apc, by BioLegend (5 mentions)
Sybr primescript rt pcr kit, by Takara Bio (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Rnalater, by Thermo Fisher Scientific (5 mentions)
Agonistic anti cd137 mab, by BPS Biosciences (4 mentions)
Anti cytokeratin mouse mab, by Abcam (4 mentions)
Mojosort human cd8 cell isolation kit, by BioLegend (4 mentions)
Anti cd3 fitc, by BioLegend (3 mentions)
Anti cd45 percp, by BioLegend (3 mentions)
Anti cd8 apc, by BioLegend (3 mentions)
Anti cd8 mouse antibody 66868 1 ig, by Proteintech (2 mentions)
Anti cd8 mouse antibody 66868 1 ig for ihc, by Proteintech (2 mentions)
Human cd3 mab okt3, by BioLegend (2 mentions)
Mitmab ab120466, by Abcam (1 mentions)
Hinokitiol, by Fujifilm (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Pe anti mouse cd31 antibody, by BioLegend (1 mentions)
Mojosort mouse anti pe nanobeads, by BioLegend (1 mentions)
11 protocols using mojosort magnet
1
Tetramer-Based Immunophenotyping of Antigen-Specific T Cells
2
Holo-Tf and Hinokitiol for Erythropoiesis
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Human holo‐Tf (208‐18971) and hinokitiol (085‐06251) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Recombinant human EPO (873999) was purchased from Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). Percoll (17‐0891‐02) was obtained from GE Healthcare (Buckinghamshire, UK). FITC‐anti‐mouse CD44 (103006), PE‐conjugated anti‐mouse Ter119 (116208), PE/Cy7‐conjugated anti‐mouse CD71 (113812), Pacific blue‐conjugated anti‐mouse CD45 (103126), biotin‐conjugated anti‐mouse CD45 antibodies (103104), 7‐aminoactinomycin D (7‐AAD; 420404), MojoSort™ Streptavidin Nanobeads (480016), and MojoSort™ Magnet (480019) were purchased from BioLegend (San Diego, CA, USA). The anti‐TfR1 monoclonal antibody R17 208.2 (sc‐65883) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Control rat IgM (14‐4341‐85), SYTO16 (S7578), and Cell‐Tak Cell and Tissue Adhesive (354240) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). RITC‐conjugated anti‐rabbit IgG antibody (SA00007‐2) was obtained from Proteintech (Rosemont, IL, USA). MitMAB (ab120466) and anti‐TfR1 antibody (ab84036) were obtained from Abcam (Cambridge, UK).
3
Purification of Mouse CD31+ Cells
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The culture medium was changed 24 h after adenovirus infection. When close to confluence, cells were digested, resuspended and washed twice with PBS. Then, cells were resuspended in 100 μL PBS, mixed with 5 μl flow cytometry antibody (PE anti-mouse CD31 Antibody, BioLegend, USA) and incubated on ice for 30 min. After this, cells were washed twice with PBS and mixed with 100 μL of the prepared 1× buffer (MojoSort™ Buffer (5×), BioLegend, USA) and 10 μl magnetic beads (MojoSort™) Mouse anti-PE Nanobeads (BioLegend, USA), then incubated on ice for 30 min. After washing twice, cell suspension was subjected to the sorting magnetic column (MojoSort™ Magnet, BioLegend, USA) for 15 min. In order to obtain higher purity sorted cells, procedures were repeated twice. Use complete medium to wash the wall of the flow cytometer to obtain the sorted positive cell suspension. Finally, positive and negative parts of the cell suspensions were identified by flow cytometric analysis.
4
Isolation of SARS-CoV-2-Specific B Cells
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Cryopreserved B cells from the nine convalescent patients were thawed in RPMI-1640 supplemented with 10% FBS tempered at 37°C; cells were centrifuged 300 x g for 8 min and resuspended for counting, and the viability of each sample was > 90%. Cells were centrifuged and resuspended in MojoSort Buffer (BioLegend, San Diego, CA, USA). S1 and RBD proteins were biotinylated using EZ-Link™ Sulfo-NHS-LC-Biotinylation Kit (Thermo Fisher Scientific); then the biotinylated proteins were incubated with the cells for 15 min at 4°C and then washed with 2 ml of Cell Staining Buffer (BioLegend, San Diego, CA, USA) and centrifuged at 300 x g for 8 min. Once labeled with SARS-CoV-2 proteins, the samples from each group of three patients were pooled. Streptavidin nanobeads (BioLegend, San Diego, CA, USA) were added to the cell pools, mixed well, and incubated for 15 min on ice. Then, 2.5 mL of MojoSort Buffer was added, and the sample tubes of each pool were placed in MojoSort Magnet (BioLegend, San Diego, CA, USA) for 5 min. The tubes were decanted without removing them from the magnet, 2.5 mL of MojoSort Buffer was added, and the last step was repeated. Finally, a positive selection of magnetically separated cells was collected and resuspended in Cell Staining Buffer; these were the enriched S1/RBD-specific B cells.
5
Immune Regulation in OP9-DL1 cDCs
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For measurement of VenusFP, UPR components, and costimulatory molecules in presence of innate immunity ligands, OP9-DL1 cDCs at 1 × 106 cells/mL were either untreated (NT) or incubated for 16 h with innate immunity ligands at the following concentrations: LPS 100 ng/mL (Invivogen, San Diego, CA, USA), Poly(I:C) 10 μg/mL (HMW, Invivogen, San Diego, CA, USA), Curdlan 50 μg/mL (Wako, Richmond, VA, USA), DMXAA 10 μg/mL (Invivogen, San Diego, CA, USA), MDP 10 μg/mL (Invivogen), and CpG (ODN 1826) 1 μM (Invivogen, San Diego, CA, USA). For costimulatory molecule analysis, OP9-DL1 cDC1s were previously sorted using Mojosort negative selection (Biolegend, San Diego, CA, USA), following manufacture’s protocol. Briefly, OP9-DL1 cDCs were stained with Sirpα-Biotin and B220-Biotin, then incubated with Mojosort Streptavidin Nanobeads (Biolegend, San Diego, CA, USA) to negatively select cDC1s using the Mojosort Magnet (Biolegend, San Diego, CA, USA). OP9-DL1 cDC1s at 1 × 106 cells/mL were either untreated (NT) or incubated for 16 h in presence of Poly(I:C) 10 or 50 μg/mL (HMW, Invivogen, San Diego, CA, USA). Cells were then harvested and used for further analysis.
6
Evaluation of Anti-CD137 Immunotherapy
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RNAlater® was purchased from Ambion, USA. TRIzol was purchased from Invitrogen, USA. DEPC was purchased from Bio Basic Inc, Canada. The SYBR® PrimeScript® RT-PCR Kit was purchased from TaKaRa, Japan for two-step RT-PCR. PCR primers were designed by TaKaRa, Japan and synthesized by Yingjun Biotechnology Co., Ltd, China. An anti-CD137 rabbit mAb (#34549) used for IHC and IF and was purchased Cell Signaling Technology (CST, USA). An IHC detection reagent (HRP, rabbit, #8114) was purchased from CST, USA. An agonistic anti-CD137 mAb (#79097) was purchased from BPS Bioscience, USA. An anti-Foxp3 rabbit mAb (#12653) used for IHC was purchased from CST, USA. Anti-CD8 mouse antibody (#66868-1-Ig) for IHC and IF was purchased from the Proteintech group, China. MojoSort™ Magnet, MojoSort™ Human CD8 Nanobeads and MojoSort™ Human CD8 Cell Isolation Kit were purchased from BioLegend, USA. An NF-κB p65 rabbit mAb (#8242) for flow cytometry and IF was purchased from CST, USA. An anti-cytokeratin mouse mAb (#ab756) used for IHC was purchased from Abcam, England. A purified anti-human CD3 mAb (OKT3, #317326) for cell incubation and anti-CD45-PerCP (#368506), anti-CD3-FITC (#300406), anti-CD8-APC (#301014) and anti-CD137-APC (#309809) antibodies for flow cytometry were purchased from BioLegend, USA.
7
Isolation and Purification of Lung Immune Cells
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Lungs were excised from CD45.1 congenic mice into DMEM media supplemented with HEPES, Penicillin, Streptomycin and L-Glutamine and digested and dissociated as above. Single cell suspensions of lung digests were combined and then a 2 X volume of ACK Lysing Buffer (Quality Biological) was added and cells were centrifuged at 350 g for 7 min at 4 °C and resuspended in PBS with 0.5% BSA (Roche) and 1% FBS (Sorting Buffer). Cells were incubated with CD11c BV650 antibody together with CD11c Mojosort Nanobeads (Biolegend), after which bead-labeled cells were purified with the Mojosort magnet (Biolegend) according to the manufacturer’s instructions. Cells were then washed and stained with sorting antibodies (Viability dye eFluor780, CD45 Alexa 700, Ly6G PE-Cy7, Siglec-F BV421, CD19 PE-Dazzle 594, CD11b BV785, MHC-II I-Ab FITC, CD64 PE, CD24 APC) at 4x107 cells/ml. Cells were washed and diluted with Sorting Buffer, centrifuged through a 1 mL layer of 100% FBS, resuspended in Sorting Buffer and passed through a 40 μm cell strainer. Cells were sorted on the FACSAria III (BD) into Advanced RPMI (Gibco) with 20% FBS.
8
Immune Checkpoint Receptor Analysis Protocol
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RNAlater® was purchased from Ambion. TRIzol was purchased from Invitrogen. DEPC was purchased from Bio Basic Inc. The SYBR®PrimeScript® RT-PCR Kit was purchased from TaKaRa for two-step RT-PCR. PCR primers were designed by TaKaRa and synthesized by Yingjun Biotechnology Co., Ltd. An anti-CD137 rabbit mAb (#34549) used for IHC and IF was purchased Cell Signaling Technology (CST, USA). An IHC detection reagent (HRP, rabbit, #8114) was purchased from CST. An agnostic anti-CD137 mAb (#79097) was purchased from BPS Bioscience. An anti-Foxp3 rabbit mAb (#12653) used for IHC was purchased from CST. An anti-CD8 mouse antibody (#66868-1-Ig) for IHC and IF was purchased from Proteintech group. MojoSort TM Magnet, MojoSort TM Human CD8 Nanobeads and MojoSort TM Human CD8 Cell Isolation Kit were purchased from BioLegend. A NF-κB p65 rabbit mAb (#8242) for ow cytometry and IF was purchased from CST. An anti-Cytokeratin mouse mAb (#ab756) used for IHC was purchased from Abcam. A puri ed anti-human CD3 mAb (OKT3, #317326) for cell incubation, and anti-CD45-PerCP (#368506), anti-CD3-FITC (#300406), anti-CD8-APC (#301014) and anti-CD137-APC (#309809) antibodies for ow cytometry were purchased from BioLegend.
9
CD137 Expression Analysis in Tumor Samples
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RNAlater® was purchased from Ambion. TRIzol was purchased from Invitrogen. DEPC was purchased from Bio Basic Inc. The SYBR® PrimeScript® RT-PCR Kit was purchased from TaKaRa for two-step RT-PCR. PCR primers were designed by TaKaRa and synthesized by Yingjun Biotechnology Co., Ltd. An anti-CD137 rabbit mAb (#34549) used for IHC and IF and was purchased Cell Signaling Technology (CST, USA). An IHC detection reagent (HRP, rabbit, #8114) was purchased from CST. An agonistic anti-CD137 mAb (#79097) was purchased from BPS Bioscience. An anti-Foxp3 rabbit mAb (#12653) used for IHC was purchased from CST. Anti-CD8 mouse antibody (#66868-1-Ig) for IHC and IF was purchased from the Proteintech group. MojoSort TM Magnet, MojoSort TM Human CD8 Nanobeads and MojoSort TM Human CD8 Cell Isolation Kit were purchased from BioLegend. An NF-κB p65 rabbit mAb (#8242) for ow cytometry and IF was purchased from CST. An anti-cytokeratin mouse mAb (#ab756) used for IHC was purchased from Abcam. A puri ed anti-human CD3 mAb (OKT3, #317326) for cell incubation and anti-CD45-PerCP (#368506), anti-CD3-FITC (#300406), anti-CD8-APC (#301014) and anti-CD137-APC (#309809) antibodies for ow cytometry were purchased from BioLegend.
10
Detailed Immunology Reagents Protocol
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RNAlater® was purchased from Ambion, USA. TRIzol was purchased from Invitrogen, USA. DEPC was purchased from Bio Basic Inc, Canada. The SYBR® PrimeScript® RT-PCR Kit was purchased from TaKaRa, Japan for two-step RT-PCR. PCR primers were designed by TaKaRa, Japan and synthesized by Yingjun Biotechnology Co., Ltd, China. An anti-CD137 rabbit mAb (#34549) used for IHC and IF and was purchased Cell Signaling Technology (CST, USA). An IHC detection reagent (HRP, rabbit, #8114) was purchased from CST, USA. An agonistic anti-CD137 mAb (#79097) was purchased from BPS Bioscience, USA. An anti-Foxp3 rabbit mAb (#12653) used for IHC was purchased from CST, USA. Anti-CD8 mouse antibody (#66868-1-Ig) for IHC and IF was purchased from the Proteintech group, China. MojoSort TM Magnet, MojoSort TM Human CD8 Nanobeads and MojoSort TM Human CD8 Cell Isolation Kit were purchased from BioLegend, USA. An NF-κB p65 rabbit mAb (#8242) for ow cytometry and IF was purchased from CST, USA. An anti-cytokeratin mouse mAb (#ab756) used for IHC was purchased from Abcam, England. A puri ed anti-human CD3 mAb (OKT3, #317326) for cell incubation and anti-CD45-PerCP (#368506), anti-CD3-FITC (#300406), anti-CD8-APC (#301014) and anti-CD137-APC (#309809) antibodies for ow cytometry were purchased from BioLegend, USA.
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