pe conjugated anti cd45, by BioLegend - Product details

1

Isolation and Characterization of Esophageal SCC Cells

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4-NQO-induced esophagus SCC were dissected, minced and digested in 2 mg/mL of collagenase I (A. G. Scientific, San Diego, CA, USA, C-2823) for 1 h. Collagenase I activity was blocked by the addition of 5 mM EDTA and incubation for 15 min. Trypsin (0.125%) (Capricorn Scientific, Ebsdorfergrund, Germany, TRY-2B10) was then added for 15 min and then the cells were rinsed in PBS supplemented with 2% FBS. All incubations were done on a rocking plate at 37 °C.
Immunostaining was performed on single cell suspension using PE-conjugated anti-CD45 (1:500, BioLegend, San Diego, CA, USA), PE-conjugated anti CD31 (1:500, BioLegend), PE-conjugated anti-CD140a (1:500, BioLegend) and Hoechst 33258 (1:10,000, Molecular Probes/Thermo Fisher Scientific, Waltham, MA, USA, H3569), during 45 min at 4 °C on a rocking plate. Living epithelial cells were selected by forward scatter, side scatter, doublets discrimination and by Hoechst dye exclusion. YFP+/Lin- cells were selected based on the expression of YFP and the exclusion of CD45, CD31, CD140a (Lin-). Control esophageal cells were selected based on the expression of APC-Cy7 EpCam (1:250, BioLegend) and the exclusion of CD45, CD31, CD140a (Lin-). Fluorescence-activated cell sorting analysis was performed using FACSAria III and FACSDiva software (BD Biosciences, San Jose, CA, USA).

Redondo J.A., Bibes R., Vercauteren Drubbel A., Dassy B., Bisteau X., Maury E, & Beck B. (2021). PER2 Circadian Oscillation Sensitizes Esophageal Cancer Cells to Chemotherapy. Biology, 10(4), 266.

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2

Multimodal analysis of engineered tumor models

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PaFs were encapsulated in either synthetic hydrogel or Matrigel in combination with mPCOs or mPCOs and BMDMs as previously described and cultured for 6 day in hPOCM supplemented with 50 ng/mL EGF and 20 ng/mL M-CSF or reduced media (Minimal media) as indicated. Single cell suspensions were prepared using 0.25% v/v Trypsin/Versene. Following neutralization with DMEM 10% v/v FBS and centrifugation cells were resuspended at 1x10⁶ cells/mL in PBS. Cells were incubated with LIVE/DEAD™ Fixable Blue Dead Cell Stain (1/1000, Thermo) at RT for 30 mins. Following washing in PBS, cells were re-suspended in CSM1 supplemented with Fc-block and subjected to surface-staining with PE conjugated anti-CD45 (1:200, BioLegend, Clone 30-F11) for 45 min on ice. Following staining, cells were washed in PBS, passed through a 70 μm filter, and re-suspended at a concentration of 1 x 10⁶cells/mL and analysed on a LSRFortessa™ X-20 flow cytometer (BD) using 355nm, 488nm, 561nm and 640nm lasers, with 450/50, 515/20, 586/15, 730/45 filter sets respectively. Cell line specific events were selected based on GFP positivity (PaF), iRFP positivity (mPCO) and CD45 positivity (BMDM) and viable cells were selected based on negativity for LIVE/DEAD blue stain.

Below C.R., Kelly J., Brown A., Humphries J.D., Hutton C., Xu J., Lee B.Y., Cintas C., Zhang X., Hernandez-Gordillo V., Stockdale L., Goldsworthy M.A., Geraghty J., Foster L., O’Reilly D.A., Schedding B., Askari J., Burns J., Hodson N., Smith D.L., Lally C., Ashton G., Knight D., Mironov A., Banyard A., Eble J.A., Morton J.P., Humphries M.J., Griffith L.G, & Jørgensen C. (2021). A microenvironment-inspired synthetic 3D model for pancreatic ductal adenocarcinoma organoids. Nature materials, 21(1), 110-119.

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3

Profiling Lung Immune Cell Populations

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Lung digests were obtained by incubation with 1 mg/mL collagenase A, and 100 ng/mL DNase (Sigma) for 1 hour. 0.5‐1 × 10(6) cells from lung digests and BAL were stained with cell surface antibodies including PerCP‐cy5.5‐conjugated anti‐F4/80, PE‐Cy7‐conjugated anti‐Ly6G, PE‐conjugated anti‐CD45, BV421‐conjugated anti‐Siglec F and APC‐conjugated anti‐CD206 (BioLegend). Analysis was performed on FACScan cytometer (Becton Dickinson). All data were analysed on FlowJo software (Tree Star).

Hu L., Chen Z., Li L., Jiang Z, & Zhu L. (2019). Resveratrol decreases CD45+CD206− subtype macrophages in LPS‐induced murine acute lung injury by SOCS3 signalling pathway. Journal of Cellular and Molecular Medicine, 23(12), 8101-8113.

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4

Multimodal analysis of engineered tumor models

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PaFs were encapsulated in either synthetic hydrogel or Matrigel in combination with mPCOs or mPCOs and BMDMs as previously described and cultured for 6 day in hPOCM supplemented with 50 ng/mL EGF and 20 ng/mL M-CSF or reduced media (Minimal media) as indicated. Single cell suspensions were prepared using 0.25% v/v Trypsin/Versene. Following neutralization with DMEM 10% v/v FBS and centrifugation cells were resuspended at 1x10⁶ cells/mL in PBS. Cells were incubated with LIVE/DEAD™ Fixable Blue Dead Cell Stain (1/1000, Thermo) at RT for 30 mins. Following washing in PBS, cells were re-suspended in CSM1 supplemented with Fc-block and subjected to surface-staining with PE conjugated anti-CD45 (1:200, BioLegend, Clone 30-F11) for 45 min on ice. Following staining, cells were washed in PBS, passed through a 70 μm filter, and re-suspended at a concentration of 1 x 10⁶cells/mL and analysed on a LSRFortessa™ X-20 flow cytometer (BD) using 355nm, 488nm, 561nm and 640nm lasers, with 450/50, 515/20, 586/15, 730/45 filter sets respectively. Cell line specific events were selected based on GFP positivity (PaF), iRFP positivity (mPCO) and CD45 positivity (BMDM) and viable cells were selected based on negativity for LIVE/DEAD blue stain.

Below C.R., Kelly J., Brown A., Humphries J.D., Hutton C., Xu J., Lee B.Y., Cintas C., Zhang X., Hernandez-Gordillo V., Stockdale L., Goldsworthy M.A., Geraghty J., Foster L., O’Reilly D.A., Schedding B., Askari J., Burns J., Hodson N., Smith D.L., Lally C., Ashton G., Knight D., Mironov A., Banyard A., Eble J.A., Morton J.P., Humphries M.J., Griffith L.G, & Jørgensen C. (2021). A microenvironment-inspired synthetic 3D model for pancreatic ductal adenocarcinoma organoids. Nature materials, 21(1), 110-119.

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5

Isolation and Characterization of Leukocytes from Murine Brain and CSF

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Before all tissue collections, mice were intracardially perfused with PBS. Brains were dissected, dissociated using a gentleMACS dissociator (Miltenyi Biotec), and loaded on a Percoll gradient (GE Healthcare) to isolate leukocytes. CSF samples were labeled without further processing. The following fluorochrome-labeled monoclonal antibodies were used according to the manufacturers’ protocols: fluorescein isothiocyanate (FITC)–conjugated anti-CD45.2, FITC-conjugated anti-TCRβ, allophycocyanin (APC)–conjugated anti-CD11b, BV421 anti-TCRβ, BV421-conjugated anti-CD45, peridinin chlorophyll protein (PerCP)–Cy5.5–conjugated anti-CXCR3, and phycoerythrin (PE)-conjugated anti-CD45 (all from BioLegend) and BD Horizon v450–conjugated anti-CD4 and Alexa Fluor 700–anti-conjugated CD45.2 (BD Biosciences). Flow cytometry analysis was performed on each sample using a BD Biosciences LSRII flow cytometer, and the acquired data were analyzed using FlowJo software (Tree Star). Gating strategies for all flow cytometry analyses are shown in fig. S8.

Kertser A., Baruch K., Deczkowska A., Weiner A., Croese T., Kenigsbuch M., Cooper I., Tsoory M., Ben-Hamo S., Amit I, & Schwartz M. (2019). Corticosteroid signaling at the brain-immune interface impedes coping with severe psychological stress. Science Advances, 5(5), eaav4111.

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6

Mouse Tumor Dissociation and Immune Profiling

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The tumor tissue of the mouse was digested with DNaseI (0.02 mg/ml, Sigma-Aldrich, St. Louis, MO, USA) and LiberaseTL (0.2 mg/ml, Roche, Basel, Switzerland) in serum-free RPMI1640 medium for 30 min. After filtered using a 70 µm cell filter, single-cell suspensions were further stained with the following fluorochrome-conjugated antibodies: PE-conjugated anti-CD45 (Biolegend) and FITC-conjugated anti-CD8 (Biolegend).

Wang J., Wu H., Chen Y., Zhu J., Sun L., Li J., Yao Z., Chen Y., Zhang X., Xia S., Chen W, & Shi T. (2021). B7-H5 blockade enhances CD8+ T-cell-mediated antitumor immunity in colorectal cancer. Cell Death Discovery, 7, 248.

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7

Multiparametric flow cytometry analysis of epithelial cells

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Immunostaining was performed on single-cell suspension using phycoerythrin (PE)–conjugated anti-CD45 (1:500; BioLegend), PE-conjugated anti CD31 (1:500; BioLegend), PE-conjugated anti-CD140a (1:500, BioLegend), and Allophycocyanin-Cyanine7 (APC-Cy7)–conjugated anti-EpCam (clone G8.8; 1:250; BioLegend) for 45 min at 4°C on a rocking plate. Living epithelial cells were selected by forward scatter, side scatter, doublets discrimination, and Hoechst dye exclusion. EpCam⁺/Lin⁻ cells were selected on the basis of the expression of EpCam and the exclusion of CD45, CD31, and CD140a (Lin⁻). For Krt8-YFP mice, YFP⁺ and YFP⁻ cells were selected within the EpCam⁺/Lin⁻ population. FACS analysis was performed using FACSAria III and FACSDiva software (BD Biosciences).

Vercauteren Drubbel A, & Beck B. (2023). Single-cell transcriptomics uncovers the differentiation of a subset of murine esophageal progenitors into taste buds in vivo. Science Advances, 9(10), eadd9135.

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Pe conjugated anti cd45

Lab products found in correlation

7 protocols using pe conjugated anti cd45

Isolation and Characterization of Esophageal SCC Cells

Multimodal analysis of engineered tumor models

Profiling Lung Immune Cell Populations

Multimodal analysis of engineered tumor models

Isolation and Characterization of Leukocytes from Murine Brain and CSF

Mouse Tumor Dissociation and Immune Profiling

Multiparametric flow cytometry analysis of epithelial cells

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