Hil 3

Manufactured by Thermo Fisher Scientific

Sourced in Germany, United States

The HIL-3 is a compact and versatile laboratory equipment designed for use in various research and analytical applications. It functions as a high-intensity light source, providing a consistent and controlled illumination environment for various experimental setups. The HIL-3 offers a range of adjustable parameters to enable precise control over the light intensity and distribution, making it a valuable tool for researchers and analysts across different fields of study.

Automatically generated - may contain errors

Lab products found in correlation

23 protocols using hil 3

Lentiviral Transduction and Myeloid Differentiation

Check if the same lab product or an alternative is used in the 5 most similar protocols

After transduction, cells were cultured in liquid culture medium (Stem Span medium supplemented with 10% v/v fetal bovine serum [FBS], 1% l-glutamine, 1% penicillin/streptomycin [P/S], mSCF [100 ng/mL], mTPO [50 ng/mL], hFlt3L [100 ng/mL], and hIL-3 [20 ng/mL]; all from PeproTech) for 3 days. To promote myeloid differentiation, medium was removed, and cells were maintained in cell expansion myeloid differentiation medium (RPMI supplemented with 10% v/v FBS, P/S, 1% l-glutamine, and mouse G-CSF [100 ng/mL]) for an additional 14 days. Transduction efficiency was evaluated by qPCR using a primers/probe set designed in the common packaging signal region (Psi) of LVs upstream of the gag start codon, as previously described.⁴⁵ (link) Mouse β-actin was used as the housekeeping gene (the list of primers and probes sequences is shown in Table S8). VCN was determined on genomic DNA extracted from transduced cells after in vitro culture for 14 days and single hematopoietic CFUs. Colonies were defined as negative CFUs when the VCN was <0.5 copy/genome and as positive when >0.5 copies/genome. Transduction efficiency was calculated as number of positive CFUs × 100/number of total CFUs.

Jofra Hernández R., Calabria A., Sanvito F., De Mattia F., Farinelli G., Scala S., Visigalli I., Carriglio N., De Simone M., Vezzoli M., Cecere F., Migliavacca M., Basso-Ricci L., Omrani M., Benedicenti F., Norata R., Rancoita P.M., Di Serio C., Albertini P., Cristofori P., Naldini L., Gentner B., Montini E., Aiuti A, & Mortellaro A. (2020). Hematopoietic Tumors in a Mouse Model of X-linked Chronic Granulomatous Disease after Lentiviral Vector-Mediated Gene Therapy. Molecular Therapy, 29(1), 86-102.

+ Open protocol

+ Expand

Differentiation and Stimulation of Human iPSC-Derived Monocytes/Macrophages

Check if the same lab product or an alternative is used in the 5 most similar protocols

For human IL-6 secretion analysis, iPSC-derived monocytes/macrophages were terminally differentiated in 50 ng/mL human M-CSF for 3-7 days. As a positive control, macrophages from peripheral blood mononuclear cells (PBMCs) were used while mouse embryonic fibroblasts were used as a negative control. PBMCs were isolated from peripheral blood of healthy volunteers using gradient centrifugation with Biocoll Separating Solution (Merck, Germany) for 40 min, 400 g. The healthy donors gave written informed consent according to the local ethical committee at Hannover Medical School. PBMCs were differentiated in RPMI1640 medium with 10% fetal calf serum, 2 mM L-glutamine, 1% penicillin-streptomycin (all Invitrogen, CA, USA) supplemented with 10 ng/mL hM-CSF and 10 ng/mL hIL-3 (Peprotech, Hamburg, Germany) for first 7 days and only 10 ng/mL hM-CSF for next 7 days. IMΦ, murine embryonic fibroblasts and PBMC-derived MΦ were cultivated in 96-well tissue culture plates for 24 h at a density of 6x10⁴ cells/well. After starvation for 24 h in X-Vivo 15 medium, iMΦ were either left unstimulated or were stimulated with 1 µg/mL LPS for another 24 h. Supernatants were collected and analyzed using the human IL-6 human uncoated ELISA Kit (Thermo Fisher, Vienna, Austria) according to the manufacturer´s instructions.

Lipus A., Janosz E., Ackermann M., Hetzel M., Dahlke J., Buchegger T., Wunderlich S., Martin U., Cathomen T., Schambach A., Moritz T, & Lachmann N. (2020). Targeted Integration of Inducible Caspase-9 in Human iPSCs Allows Efficient in vitro Clearance of iPSCs and iPSC-Macrophages. International Journal of Molecular Sciences, 21(7), 2481.

+ Open protocol

+ Expand

iPSC-Derived Microglial Characterization

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

iPSCs were first differentiated into hematopoietic progenitor cells following manufacturer’s instructions using a commercially available kit (StemCell Technologies #05310) as described before (Andreone et al., 2020 (link)). HPCs positive for identity markers CD34, CD43, and CD45 were transferred to a plate containing primary human astrocytes and co-cultured using media C adapted from a previous study (Pandya et al., 2017 (link)). Once floating cells in co-culture are predominantly (>80%) mature microglia, cells were plated for 3–4 days prior to experiments. Full characterization of human iPSC-derived microglia and additional details on the differentiation protocol has been published elsewhere (Andreone et al., 2020 (link)). All the experiments using GRN^+/+ and GRN^−/− iMG were performed in IMDM (Gibco) media supplemented with 10% defined FBS (Gibco), 1% Penicillin/Streptomycin (Gibco), 20 ng/mL of hIL3 (Peprotech), 20 ng/mL of hGM-CSF (Peprotech) and 20 ng/mL of hM-CSF (Peprotech) (referred to as “C+++ Media”). Immunostainings of PGRN were conducted using the anti-progranulin goat polyclonal antiserum (R&D Systems # AF2420, 1:250) to verify the absence of immunoreactivity in knockout iMG.

Logan T., Simon M.J., Rana A., Cherf G.M., Srivastava A., Davis S.S., Yoon Low R.L., Chiu C.L., Fang M., Huang F., Bhalla A., Llapashtica C., Prorok R., Pizzo M.E., Calvert M.E., Sun E.W., Hsiao-Nakamoto J., Rajendra Y., Lexa K.W., Srivastava D.B., van Lengerich B., Wang J., Robles-Colmenares Y., Kim D.J., Duque J., Lenser M., Earr T.K., Nguyen H., Chau R., Tsogtbaatar B., Ravi R., Skuja L.L., Solanoy H., Rosen H.J., Boeve B.F., Boxer A.L., Heuer H.W., Dennis M.S., Kariolis M.S., Monroe K.M., Przybyla L., Sanchez P.E., Meisner R., Diaz D., Henne K.R., Watts R.J., Henry A.G., Gunasekaran K., Astarita G., Suh J.H., Lewcock J.W., DeVos S.L, & Di Paolo G. (2021). Rescue of a lysosomal storage disorder caused by Grn loss-of-function with a brain penetrant progranulin biologic. Cell, 184(18), 4651-4668.e25.

+ Open protocol

+ Expand

Colony-Forming Cell Assay Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

For CFC assays, 1,000–1,200 FACS-purified CD34 subpopulations were seeded into MethoCult H4435 (STEMCELL Technologies) or H4230 supplemented with human interleukin (hIL)-3, IL-5, G-CSF, stem cell factor (SCF), thyroperoxidase (TPO), and granulocyte-macrophage colony-stimulating factor (GM-SCF) (all PeproTech), each at 100 μg/mL, as well as erythropoietin (EPO) (Amgen, Thousand Oaks, CA, USA) at 4 U/mL for the large-scale clinical grade experiments according to our established clinical protocols.³⁹ ^,⁴⁸ (link) Colonies were scored after 12–14 days, discriminating CFU-granulocyte (CFU-G), CFU-macrophage (CFU-M), granulocyte-macrophage (CFU-GM), and burst forming unit-erythrocyte (BFU-E). Colonies consisting of erythroid and myeloid cells were scored as CFU-MIX.

Radtke S., Pande D., Cui M., Perez A.M., Chan Y.Y., Enstrom M., Schmuck S., Berger A., Eunson T., Adair J.E, & Kiem H.P. (2020). Purification of Human CD34+CD90+ HSCs Reduces Target Cell Population and Improves Lentiviral Transduction for Gene Therapy. Molecular Therapy. Methods & Clinical Development, 18, 679-691.

+ Open protocol

+ Expand

Transduction of Patient-Derived AML Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Patient-derived AML samples were obtained from The Ohio State Comprehensive Cancer Center and used according to the approved IRB protocol 16-9037. Cells were plated on irradiated mono-layers of HS27 cells (21 (link)) and cultured in Stemspan (Stem Cell Technology) supplemented with 10% FBS, 100ng/ml hSCF (Peprotech), 100ng/ml hFLT3 ligand (Peprotech), 20ng/ml hIL-3 (Peprotech), 20ng/ml hIL-6 (Peprotech), 20ng/ml G-CSF (Peprotech). 500,000 cells were transduced with pLKO.1 GFP lentiviral shRNA vectors and evaluated for GFP expression every 3 days for 12 days after staining with human CD45 APC-Cy7 (BD Biosciences) and Propidium Iodide (PI) by flow cytometry.

Di Marcantonio D., Martinez E., Sidoli S., Vadaketh J., Nieborowska-Skorska M., Gupta A., Meadows J.M., Ferraro F., Masselli E., Challen G.A., Milsom M.D., Scholl C., Fröhling S., Balachandran S., Skorski T., Garcia B.A., Mirandola P., Gobbi G., Garzon R., Vitale M, & Sykes S.M. (2017). PKC epsilon is a Key Regulator of Mitochondrial Redox Homeostasis in Acute Myeloid Leukemia. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(3), 608-618.

+ Open protocol

+ Expand

Quantifying Hematopoietic Progenitor Colonies

Check if the same lab product or an alternative is used in the 5 most similar protocols

For granulocyte-macrophage colonies 30 000 fresh BM cells from steady state mice were plated in methylcellulose medium (M3231; StemCell Technologies) containing 50 ng/mL mSCF, 10 ng/mL murine interleukin 3 (IL-3; PreproTech), 10 ng/mL human IL-6 (PreproTech), 100 IU penicillin and 100 μg streptomycin (P/S; Invitrogen) in 35-mm petri dishes. For erythroid colonies 150 000 BM cells were plated in methylcellulose medium (M3236; StemCell Technologies), supplemented with 50 ng/mL mSCF, 50 ng/mL hTPO and 5 U/mL human erythropoietin (Epo; Apoteket Farmaci) and P/S (Invitrogen). Colony numbers were scored after 12 days of culture. For total colony capacity of cultured CD34⁺ human cells, 250–300 cells were plated in 35-mm petri dishes in methylcellulose medium (M4230; StemCell Technologies) containing hSCF (25 ng/mL), GM-CSF (50 ng/mL, R&D), hIL-3 (25 ng/mL, PreproTech), hEPO (5 U/mL, Apoteket Farmaci, Lund, Sweden), and P/S (Invitrogen). Total colony number was scored after 12–14 days of culture. Cells were incubated at 37 °C in 5% CO₂.

Rörby E., Billing M., Dahl M., Warsi S., Andradottir S., Miharada K., Siva K., Jönsson J.I., Blank U., Karlsson G, & Karlsson S. (2017). The stem cell regulator PEDF is dispensable for maintenance and function of hematopoietic stem cells. Scientific Reports, 7, 10134.

+ Open protocol

+ Expand

Immune Cell Activation Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell cultures were stimulated with medium, 5 μg/ml R848, 5 μg/ml CpG2216, 1 μg/ml LPS, 5 μg/ml poly(I:C), or 5 μg/ml R848 + 5 μg/ml HMW poly(I:C) (InvivoGen) directly in the 24 well plates used for differentiation. Half of the culture medium was replaced by fresh one supplemented with the TLR agonists and 200ng/ml hFLT3L, 50ng/ml hIL3 (Peprotech). Brefeldin A (Sigma) was added at a final concentration of 10 μg/ml after 2h (6h time point) or 14h (16h time point) of stimulation. For the analysis of activation markers, cells were stimulated for 16h without Brefeldin A.

Balan S., Arnold-Schrauf C., Abbas A., Couespel N., Savoret J., Imperatore F., Villani A.C., Vu Manh T.P., Bhardwaj N, & Dalod M. (2018). Large-Scale Human Dendritic Cell Differentiation Revealing Notch-Dependent Lineage Bifurcation and Heterogeneity. Cell Reports, 24(7), 1902-1915.e6.

+ Open protocol

+ Expand

Measuring Influenza Virus Entry and Protein Synthesis in PBMCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

Infection of primary human PBMCs, isolated subpopulations of PBMCs and PBMCs-derived macrophages were carried out at a cell density of 5 x 10⁶ cells/ml in RPMI supplemented with 0. 1% BSA, 1 % glutamine and pen/strep using individual Teflon vials with rounded interior (Savillex, 200-015-20). Recombinant influenza viruses were added at multiplicity of infection (MOI) 3, 0.3 and 0.03 FFU/cell. hIL-3 (Peprotech, #200-03-100UG) was added in a final concentration of 10 ng/ml. Infected cells were incubated for 6-8 h at 37°C, 5 % CO₂. In these experiments, we studied the ability of the viruses to enter the cells and to initiate synthesis of viral proteins as measured by the detection of NP production via flow cytometry.

Dorna J., Kaufmann A., Bockmann V., Raifer H., West J., Matrosovich M, & Bauer S. (2022). Effects of Receptor Specificity and Conformational Stability of Influenza A Virus Hemagglutinin on Infection and Activation of Different Cell Types in Human PBMCs. Frontiers in Immunology, 13, 827760.

+ Open protocol

+ Expand

Erythroid Differentiation of CD34+ Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Transduced CD34⁺ cells were transferred into erythroid culture. The in vitro erythroid differentiation technique used is based on a 3-phase protocol adapted from Giarratana et al.²⁸ (link) The basic erythroid medium was IMDM (Life Technologies), 1 × LGlut-Pen-Strep, 10% BSA, 40 μg/mL inositol, 10 μg/mL folic acid, 1.6 μM monothioglycerol, 120 μg/mL transferrin, and 10 μg/mL insulin (all from Sigma-Aldrich, St. Louis, MO, USA). During the first phase (6 days), the cells were cultured in the presence of 1 × 10⁻⁶ M hydrocortisone (Sigma-Aldrich), 100 ng/mL hSCF, 5 ng/mL hIL-3 (Peprotech), and 3 IU/mL erythropoietin (Epo; Janssen Pharmaceuticals). In the second phase (3 days), the cells were transferred onto a stromal cell layer (MS-5, murine stromal cell line,²⁹ (link) provided by Gay Crooks, UCLA, Los Angeles, CA, USA) with the addition of only Epo (3 IU/mL) to basic erythroid medium. At day 11, all the cytokines were removed from the medium, and the cells were co-cultured on the MS-5 stromal layer until day 14, when they were collected to extract genomic DNA and RNA.

Masiuk K.E., Zhang R., Osborne K., Hollis R.P., Campo-Fernandez B, & Kohn D.B. (2019). PGE2 and Poloxamer Synperonic F108 Enhance Transduction of Human HSPCs with a β-Globin Lentiviral Vector. Molecular Therapy. Methods & Clinical Development, 13, 390-398.

+ Open protocol

+ Expand

Differentiation and Maintenance of Murine and Human Microglia

Check if the same lab product or an alternative is used in the 5 most similar protocols

Primary BMDMs were harvested from 3–6 month old male and female mice. Cells were cultured in 10mL RPMI-1640 (ThermoFisher) + 10% Hyclone FBS (GE Healthcare) + Penicillin-Streptomycin (ThermoFisher) at 37°C and 5% CO_2. Expi293F cells (ThermoFisher) were maintained according to the manufacturer’s specifications in Expi293 Expression medium (ThermoFisher #A1435101). Human induced-pluripotent stem cell lines (hiPSCs) were generated from a female clone from ThermoFisher (#A18945) and routinely passaged as clumps onto Geltrex (Thermo #A1413302)-coated plates with mTeSR1 media (StemCell Technologies #85850) according to manufacturer’s instructions. All the experiments using GRN^+/+ and GRN^−/− iMG were performed in IMDM (Gibco) media supplemented with 10% defined FBS (Gibco), 1% Penicillin/Streptomycin (Gibco), 20 ng/mL of hIL3 (Peprotech), 20 ng/mL of hGM-CSF (Peprotech) and 20 ng/mL of hM-CSF (Peprotech) (referred to as “C+++ Media”) and maintained at 37°C and 5% CO₂. Full characterization of human iPSC-derived microglia and additional details on the differentiation protocol has been published elsewhere (Andreone et al., 2020 (link)).

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!