Polypropylene facs tube, by BD - Product details

1

Multiparameter Flow Cytometry Staining Protocol

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For surface staining, 5×10⁶ erythrolyzed and washed cells in 50 µl of FACS buffer (PBS, 2% FCS, 0.05% NaN₃) were stained with prepared antibody multi-mixes (obtained from eBioscience, BD Pharmingen or BioLegend) in the presence of Fc receptor blocking antibody (clone 24G.2) to prevent non-specific binding of Fc receptors. Cells were incubated at 4°C for 40 minutes. Stained cells were washed two times with 200 µl of FACS buffer. For biotinylated antibodies, streptavidin-conjugated fluorochromes were added at a dilution of 1/100 and further incubated for 10 minutes at room temperature and then washed 3 times with FACS buffer. Cells were acquired using the CyAn ADP flow cytometer (Beckman Coulter) within 2 hours. Data were analyzed using FlowJo (Tree Star, Inc). Flowjo was used for graphical representation. For Annexin V staining, after surface staining was completed, cells were washed 2 times and resuspended in Annexin V Binding Buffer (BioLegend) at a concentration of 1×10⁶ cells/ml. 100 µl of the cell suspension was filtered through a 0.2 µm filter into polypropylene FACS tubes (BD) and 5 µl of Annexin V-Pacific Blue (BioLegend) was added. Cells were incubated for 15 minutes at room temperature in the dark. 400 µl of Annexin V Binding Buffer was added to the tube just prior to analysis.

Ng D.H., Skehel J.J., Kassiotis G, & Langhorne J. (2014). Recovery of an Antiviral Antibody Response following Attrition Caused by Unrelated Infection. PLoS Pathogens, 10(1), e1003843.

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2

Mammary Gland Cell Isolation and FACS Analysis

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Isolated mammary glands were minced and digested as above. Red blood cells were lysed with red blood cell lysis buffer (Sigma-Aldrich) by manufacturer's instructions. Samples were treated with 100 µg/mL DNase (Sigma-Aldrich) for 2 min at 37°C and filtered using 30-µm filters. Cells were counted, resuspended up to 10⁷ cells/mL per 90 µL FACS buffer (PBS + 5% CS), and treated with FcR blocking reagent (1:10; Miltenyi Biotec 130-092-575) for 30 min on ice. Cell suspensions were transferred to polypropylene FACS tubes (BD Biosciences) and either left unstained, stained with isotype control (1:50; Biolegend 400511), or stained with CD326-APC (EpCAM, 1:50; Biolegend 118214) for 30 min on ice. Cells were washed with 1 mL of FACS buffer and sorted using a Beckman Coulter MoFlo XDP at the UCSF Parnassus Flow Cytometry Core.

Rudnick J.A., Monkkonen T., Mar F.A., Barnes J.M., Starobinets H., Goldsmith J., Roy S., Bustamante Eguiguren S., Weaver V.M, & Debnath J. (2021). Autophagy in stromal fibroblasts promotes tumor desmoplasia and mammary tumorigenesis. Genes & Development, 35(13-14), 963-975.

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3

Aortic Cell Composition Analysis

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Cellular composition of aortas was determined in the Cell Phenotyping Core of the UCSD PPG on Role of Immune Mechanisms in Inflammation and Atherosclerosis, under the direction of K. Ley, using established techniques³⁷. In brief, 6 aortas from 16-week chow-fed Ldlr^−/−, 6 from HFD-fed Ldlr^−/− and 5 from Ldlr^−/−/E06-scFv mice were dissected following heparin PBS perfusion and adventitia carefully removed. The intact aortas were incubated for one hour with an Aorta Dissociation Enzyme stock solution and single cell suspensions prepared from the digested aorta by shearing the aortas apart and passing cells through a 70μm cell strainer into 5ml polypropylene FACS tubes (BD Falcon). The cells were pelleted by centrifugation (400×g, 5 minutes, 4°C), resuspended in 1ml of FACS buffer (PBS supplemented with 1% BSA and 0.05% NaN₃), counted and assessed for viability using trypan blue in a hemocytometer. Cells were stained on ice for 30 min with the panel of antibodies below, washed twice with FACS buffer and then analyzed at La Jolla Institute for Allergy and Immunology using a FACSAria analyzer. Anti-CD45 antibody and fixable live-dead cell stain (Invitrogen, Molecular Probes) was added to all samples to allow for gating of live CD45+ leukocytes and cells were sorted with the panel of antibodies listed in Table in Supplemental Information.

Que X., Hung M.Y., Yeang C., Gonen A., Prohaska T.A., Sun X., Diehl C., Määttä A., Gaddis D.E., Bowden K., Pattison J., MacDonald J.G., Ylä-Herttuala S., Mellon P.L., Hedrick C.C., Ley K., Miller Y.I., Glass C.K., Peterson K.L., Binder C.J., Tsimikas S, & Witztum J.L. (2018). Oxidized Phospholipids are Proinflammatory and Proatherogenic in Hypercholesterolemic Mice. Nature, 558(7709), 301-306.

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4

Quantifying S-Phase Cells in PEG-VS Hydrogels

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To identify cells in S phase 5-Iodo-2′-deoxyuridine (IdU, Sigma) was added to organoid culture medium at a final concentration of 25 μM and incubated for 30 mins at 37°C. Gels were then dissociated immediately or fixed in 4% paraformaldehyde (PFA) (Thermo Fisher scientific) for 60 min at 37°C followed by 2x wash in PBS (Fisher) and storage at 4°C until required. Both fixed and un-fixed PEG-VS hydrogels were dissolved using SrtA (Supplemental materials and methods). The resultant solution was transferred to a gentleMACS C-Tube (Miltenyi) containing 0.5 mg/ml Dispase II (Sigma), 0.2 mg/ml Collagenase IV (Gibco) and 0.2 mg/ml DNase (Gibco) made up to 5 mL in PBS and processed using the gentleMACS Octo Dissociator (with Heaters) (Miltenyi) at 37°C for 50min using a custom program adapted from C.Tape^{62 (link)}. C-tube was spun at 1000g for 1 min to collect all cellular material in the bottom of the tube. Cells were washed in 5 mL of PBS and passed through a 70-μm filter (BD) into polypropylene FACS tubes (BD Falcon) to remove residual cell clusters.

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

Quantifying S-Phase Cells in PEG-VS Hydrogels

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To identify cells in S phase 5-Iodo-2′-deoxyuridine (IdU, Sigma) was added to organoid culture medium at a final concentration of 25 μM and incubated for 30 mins at 37°C. Gels were then dissociated immediately or fixed in 4% paraformaldehyde (PFA) (Thermo Fisher scientific) for 60 min at 37°C followed by 2x wash in PBS (Fisher) and storage at 4°C until required. Both fixed and un-fixed PEG-VS hydrogels were dissolved using SrtA (Supplemental materials and methods). The resultant solution was transferred to a gentleMACS C-Tube (Miltenyi) containing 0.5 mg/ml Dispase II (Sigma), 0.2 mg/ml Collagenase IV (Gibco) and 0.2 mg/ml DNase (Gibco) made up to 5 mL in PBS and processed using the gentleMACS Octo Dissociator (with Heaters) (Miltenyi) at 37°C for 50min using a custom program adapted from C.Tape^{62 (link)}. C-tube was spun at 1000g for 1 min to collect all cellular material in the bottom of the tube. Cells were washed in 5 mL of PBS and passed through a 70-μm filter (BD) into polypropylene FACS tubes (BD Falcon) to remove residual cell clusters.

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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6

Aortic Cell Composition Analysis

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Cellular composition of aortas was determined in the Cell Phenotyping Core of the UCSD PPG on Role of Immune Mechanisms in Inflammation and Atherosclerosis, under the direction of K. Ley, using established techniques³⁷. In brief, 6 aortas from 16-week chow-fed Ldlr^−/−, 6 from HFD-fed Ldlr^−/− and 5 from Ldlr^−/−/E06-scFv mice were dissected following heparin PBS perfusion and adventitia carefully removed. The intact aortas were incubated for one hour with an Aorta Dissociation Enzyme stock solution and single cell suspensions prepared from the digested aorta by shearing the aortas apart and passing cells through a 70μm cell strainer into 5ml polypropylene FACS tubes (BD Falcon). The cells were pelleted by centrifugation (400×g, 5 minutes, 4°C), resuspended in 1ml of FACS buffer (PBS supplemented with 1% BSA and 0.05% NaN₃), counted and assessed for viability using trypan blue in a hemocytometer. Cells were stained on ice for 30 min with the panel of antibodies below, washed twice with FACS buffer and then analyzed at La Jolla Institute for Allergy and Immunology using a FACSAria analyzer. Anti-CD45 antibody and fixable live-dead cell stain (Invitrogen, Molecular Probes) was added to all samples to allow for gating of live CD45+ leukocytes and cells were sorted with the panel of antibodies listed in Table in Supplemental Information.

Que X., Hung M.Y., Yeang C., Gonen A., Prohaska T.A., Sun X., Diehl C., Määttä A., Gaddis D.E., Bowden K., Pattison J., MacDonald J.G., Ylä-Herttuala S., Mellon P.L., Hedrick C.C., Ley K., Miller Y.I., Glass C.K., Peterson K.L., Binder C.J., Tsimikas S, & Witztum J.L. (2018). Oxidized Phospholipids are Proinflammatory and Proatherogenic in Hypercholesterolemic Mice. Nature, 558(7709), 301-306.

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7

Multicolor Flow Cytometry of Thawed PBMCs

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Two million thawed PBMCs were stained with fixable viability dye eFluor 506 (eBioscience) and with anti-human CD3, CD4, CD8, CD19, CD14, and CD45RO (see Supplemental Table I for Ab details) for 30 min at 4°C, protected from light. After two washes in PBS, cells were fixed and permeabilized with an NF Fixation and Permeabilization Buffer Set (BioLegend), according to the manufacturer’s instructions. Subsequently, cells were stained with anti-human Bcl2 for 30 min at room temperature. After two washes in PBS, cells were resuspended in 100 μl of PBS containing 0.5% FBS and 2 mM EDTA (pH 8; FACS Buffer), transferred into a 5-ml polypropylene FACS tube (BD Biosciences), and stored at 4°C protected until acquisition. FACS acquisition was performed on a BD LSR II cell analyzer (BD Biosciences), and gating was performed with FlowJo (version 10.1).

Burel J.G., Lindestam Arlehamn C.S., Khan N., Seumois G., Greenbaum J.A., Taplitz R., Gilman R.H., Saito M., Vijayanand P., Sette A, & Peters B. (2018). Transcriptomic Analysis of CD4+ T Cells Reveals Novel Immune Signatures of Latent Tuberculosis. Journal of immunology (Baltimore, Md. : 1950), 200(9), 3283-3290.

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8

Isolation and Culture of Haploid ES Cells

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Following culture of FACS-purified ahaES cells at 37 °C in humidified 5% (v/v) CO₂ in air, cell suspensions were prepared as previously described^{13 (link),61 (link)}. Briefly, cells were washed with DMEM medium followed by calcium-free PBS and incubated with trypsin/EDTA for 3 min at 37 °C. Trypsinization was quenched by the addition of 5 ml ES/DMEM (DMEM supplemented with 5% [v/v] FCS/LIF^{13 (link)}) and cells dissociated by gentle pipetting. Single-cell suspensions were pelleted by centrifugation (221g, 5 min) and resuspended in fresh ES/DMEM medium. Single-cell ahaES cell suspensions were placed on ice and used immediately for micromanipulation. In some cases, haploid cells were enriched by FACS sorting immediately prior to micromanipulation. Cell aggregates were removed by passing suspensions though a 50 μm cell strainer (Falcon) into a polypropylene FACS tube (BD). To avoid Hoechst toxicity, we employed SSC and FSC as FACS Aria parameters for haploid and diploid population separation. Enriched haploid ES cells were collected into an ice-cold FACS tube containing 1 ml ES/DMEM supplemented with serum and immediately used for micromanipulation. G1 cell selection was further attempted by selecting smaller cells as nucleus donors.

Santini L., Halbritter F., Titz-Teixeira F., Suzuki T., Asami M., Ma X., Ramesmayer J., Lackner A., Warr N., Pauler F., Hippenmeyer S., Laue E., Farlik M., Bock C., Beyer A., Perry A.C, & Leeb M. (2021). Genomic imprinting in mouse blastocysts is predominantly associated with H3K27me3. Nature Communications, 12, 3804.

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Polypropylene facs tube

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8 protocols using polypropylene facs tube

Multiparameter Flow Cytometry Staining Protocol

Mammary Gland Cell Isolation and FACS Analysis

Aortic Cell Composition Analysis

Quantifying S-Phase Cells in PEG-VS Hydrogels

Quantifying S-Phase Cells in PEG-VS Hydrogels

Aortic Cell Composition Analysis

Multicolor Flow Cytometry of Thawed PBMCs

Isolation and Culture of Haploid ES Cells

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