Phytohemagglutinin pha m

Manufactured by Merck Group

Sourced in United States, United Kingdom

Phytohemagglutinin (PHA-M) is a plant-derived lectin that is commonly used as a mitogen in cell culture applications. It functions by stimulating lymphocyte proliferation, which is a key step in various immunological assays and research studies.

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Lab products found in correlation

Lymphoprep, by Abbott (2 mentions) Rpmi 1640, by Thermo Fisher Scientific (2 mentions) L glutamine, by Merck Group (2 mentions) Peptide array, by BEI Resources (1 mentions) Ficoll paque plus, by Cytiva (1 mentions) Brdu elisa, by Roche (1 mentions) 96 well plate, by Thermo Fisher Scientific (1 mentions) Zymosan, by InvivoGen (1 mentions) Non essential amino acid (neaa), by Merck Group (1 mentions) Resiquimod, by InvivoGen (1 mentions) Biocoll separating solution, by Harvard Bioscience (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions) Sodium pyruvate, by Merck Group (1 mentions) Cytochalasin b, by Merck Group (1 mentions) Methylene blue, by Merck Group (1 mentions) Ferret ifn γ elisa development kit alp, by Mabtech (1 mentions) Fixation buffer, by BD (1 mentions) Cytomics fc500, by Beckman Coulter (1 mentions)

9 protocols using phytohemagglutinin pha m

Tuberculosis Antigen Peptide Pools Protocol

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Initial optimization experiments were conducted using pooled, overlapping
15- or 16-mer peptides corresponding to the sequences of CFP-10, ESAT-6, and
TB10.4 (BEI Resources, NIAID, NIH: Peptide Array, M.
tuberculosis CFP-10 Protein, NR-34825; Peptide Array, M.
tuberculosis ESAT-6 Protein, NR-34824; Peptide Array, M.
tuberculosis TB10.4 Protein, NR-34826). 18-mer peptides
(overlapping by 11 amino acids) corresponding to the 60 Mtb Ags listed in Table 1 were synthesized on a 10mg scale
using Fmoc chemistry (Synthetic Biomolecules, San Diego, CA) according to the
H37Rv genome sequence. Peptides were pooled by Ag (10 – 121 peptides per
pool, with a median of 39 peptides per pool); peptide pools were numbered
consecutively according to Rv gene numbers (Table 1). After pooling the peptides for each Ag, pools were
lyophilized, followed by reconstitution in DMSO at a final centration of 1mg/ml
per peptide. Peptide pools were further diluted in RPMI-1640 medium supplemented
with 2mM L-glutamine, 100U/ml penicillin, and 100µg/ml streptomycin, to
prepare working stocks for use in the stimulation assays described below.
Phytohemagglutinin-M (PHA; Sigma-Aldrich) was used a positive control.

Whatney W.E., Gandhi N.R., Lindestam Arlehamn C.S., Nizam A., Wu H., Quezada M.J., Campbell A., Allana S., Kabongo M.M., Khayumbi J., Muchiri B., Ongalo J., Tonui J., Sasser L.E., Fergus T.J., Ouma G.S., Ouma S.G., Beck A.A., Mulligan M.J., Oladele A., Kaushal D., Cain K.P., Waller L., Blumberg H.M., Altman J.D., Ernst J.D., Rengarajan J, & Day C.L. (2018). A high throughput whole blood assay for analysis of multiple antigen-specific T cell responses in human Mycobacterium tuberculosis infection. Journal of immunology (Baltimore, Md. : 1950), 200(8), 3008-3019.

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PBMC isolation and stimulation assay

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Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy consenting adult donors using a Ficoll density gradient (Ficoll Paque Plus, Cytiva). 6 donors participated in this study, and 60ml of blood was collected per donor. Blood was centrifuged at 1000g for 15 minutes in 50ml LeucoSep tubes. 1-1.2 million cells were seeded per well in a 24 well plate in RPMI media with 10% FBS and 1% Penicillin/Streptomycin per well in a total volume of 1ml. For PHA-stimulation 5µg/ml of Phytohemagglutinin-M (PHA) (Sigma 11082132001) was added to PBMCs in culture to stimulate T cell proliferation and activation. PBMCs were suspended in 1ml total volume which consisted of 800µl of media and 200µl of a treatment solution or a vehicle control. Treatment solutions added to PHA-stimulated cells included acAF, AF-EV preparations, and EV-depleted supernatant.

del Rivero T., Milberg J., Bennett C., Mitrani M.I, & Bellio M.A. (2022). Human amniotic fluid derived extracellular vesicles attenuate T cell immune response. Frontiers in Immunology, 13, 977809.

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Immunogenicity and Immunosuppression Capacity of ASCs

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For immunogenicity and immunosuppression capacity studies, peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat samples and frozen as previously described [8 (link)]. The buffy coat samples were obtained from the Finnish Red Cross Blood Service, and the study was conducted in accordance with the Declaration of Helsinki 1975, revised in Hong Kong 1989. The immunogenicity of ASCs was studied using a one-way mixed lymphocyte reaction (MLR) as previously described [8 (link)]. Briefly, irradiated ASCs at a density of 62,500 cells/cm² at passage 4 or 5 were cocultured with PBMCs at a density of 625,000 cells/cm² in 96-well plates (Nunc) for 5 days in HPL medium. For nonproliferative control samples, PBMCs were irradiated to prevent cell proliferation, and for proliferative control samples, PBMCs were cultured with 10 µg/ml phytohemagglutinin-M (PHA; Sigma-Aldrich) to enhance proliferation. The proliferation of PBMCs was measured with BrdU ELISA (Roche Applied Science, Penzberg, Germany).

Juntunen M., Heinonen S., Huhtala H., Rissanen A., Kaprio J., Kuismanen K., Pietiläinen K.H., Miettinen S, & Patrikoski M. (2021). Evaluation of the effect of donor weight on adipose stromal/stem cell characteristics by using weight-discordant monozygotic twin pairs. Stem Cell Research & Therapy, 12, 516.

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Engineered CAR T cells from GBM patients

Cited 2 times

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CAR T cells were engineered starting from GBM patients’ PBMCs (as approved by the Institutional Review Board) which were separated by a density gradient (Lymphoprep, Alere Technologies AS, Oslo, Norway; #1114545) and then plated in RPMI 1640 (Gibco; #52400-017) with 1% FBS, 1% glutamine and 1% penicillin-streptomycin. Non-adherent cells were pre-stimulated for 48 h in 7 µg/mL Phytohemagglutinin (PHA-M, Sigma-Aldrich; #L8902) at a concentration of 1 × 10⁶ cells/mL in a culture medium made by RPMI 1640 supplemented with 10% heat-inactivated defined FBS (HyClone Laboratories, Utah, USA; #SH30070.03), 500 UI/mL recombinant human Interleukin-2 (rhIL-2, Proleukin, Clinigen Healthcare Ltd, Staffordshire DE14 2WW, UK; #801313AY). Isolated T lymphocytes were transduced by a retroviral vector bearing second-generation anti-GD2 CAR and GFP control vector^{11 (link),12 (link)}.

Chiavelli C., Prapa M., Rovesti G., Silingardi M., Neri G., Pugliese G., Trudu L., Dall’Ora M., Golinelli G., Grisendi G., Vinet J., Bestagno M., Spano C., Papapietro R.V., Depenni R., Di Emidio K., Pasetto A., Nascimento Silva D., Feletti A., Berlucchi S., Iaccarino C., Pavesi G, & Dominici M. (2024). Autologous anti-GD2 CAR T cells efficiently target primary human glioblastoma. NPJ Precision Oncology, 8, 26.

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PBMC Isolation and Stimulation Protocol

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Blood samples were collected in tubes with lithium heparin (Vacutainer®) and diluted with an equal volume of warm PBS (Gibco, Invitrogen, Massachusetts). PBMCs were isolated by centrifuging at 800 g for 20 min at 18–20℃ on Biocoll separating solution (Biochrom AG, Germany). PBMCs were washed three times, and the cell pellet was resuspended in complete medium [RPMI 1640 with HEPES 25 mM and l‐Glutamine (Gibco, Life Technologies Ltd, UK), supplemented with 10 ml/L Penicillin‐Streptomycin USA, 50 μl/L 1 M β‐mercaptoethanol, 20 ml/L l‐Glutamine plus MEM Vitamin, 20 ml/L Non‐essential Amino Acid, Sodium Pyruvate and 10% heat‐inactivated FBS (all from Sigma‐Aldrich, Germany)]. The suspension was seeded in a flat‐bottom 48‐well tissue plate (Corning Incorporated, Costar, New York), with 5 × 10⁵ viable cells per well (500 μl). PBMCs were cultured in duplicates either with complete medium alone (unstimulated control) or with one of the following stimulants: 4 μg/ml Resiquimod (R848), 10 μg/ml Zymosan (InvivoGen, France), 10 μg/ml Phytohemagglutinin (PHA‐M), 20 μg/ml Polyinosinic–polycytidylic acid potassium salt (Poly I:C), (Sigma‐Aldrich, Germany), at 37℃, 5% CO₂. Cultures were harvested after 48 h and, after centrifugation at 600 g for 5 min, supernatants were stored at −80℃ until analysis.

Maurer D.J., Liu C., Xepapadaki P., Stanic B., Bachert C., Finotto S., Gao Y., Graser A., Jartti T., Kistler W., Kowalski M., Lukkarinen H., Pasioti M., Tan G., Villiger M., Zhang L., Zhang N., Akdis M., Papadopoulos N.G, & Akdis C.A. (2021). Physical activity in asthma control and its immune modulatory effect in asthmatic preschoolers. Allergy, 77(4), 1216-1230.

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Cytogenetic Stability Biomarker Protocol

Cited 1 time

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Cytogenetic stability biomarkers were evaluated based on the cytokinesis-block technique established by Fenech [28 (link)]. Briefly, 5 mL of blood was obtained from each participant by venipuncture, which was cultured by duplicate in 15 mL Falcon^® tubes (Corning, Glendale, AZ, USA) with 6.3 mL of RPM1-1640 culture medium (Sigma R-8758) supplemented with non-essential amino acids (Sigma M-7145, St. Louis, MO, USA), L-Glutamine (Sigma G-6392, St. Louis, MO, USA), and 0.2 mL of phytohemagglutinin PHA-M (Sigma L-8902, St. Louis, MO, USA). The tubes were incubated at 37 °C for 44 h, stopping proliferation by adding 3µL/mL of cytochalasin-B (Sigma C-6762, St. Louis, MO, USA); after that, the cells were incubated for another 24 h at 37 °C. The lymphocytes were recovered from the culture with a solution of methanol and acetic acid (3:1). After washing, the lymphocytes were stained with eosin (Sigma 318906, St. Louis, MO, USA) and methylene blue (Sigma 03978, St. Louis, MO, USA) and observed under a compound microscope (100×) [32 (link)].

Ruiz-Ruiz B., Torres-Bugarin O., Zúñiga-Violante E., Casillas-Figueroa F., Luna-Vázquez-Gómez R., Campos Gallegos V., Ruiz-Arellano A.E, & Arellano-García M.E. (2023). Genomic Instability and Cytotoxicity Evaluation of Two Communities Exposed to Pesticides in the Mexicali Valley by the L-CBMN Assay. Toxics, 11(10), 807.

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Influenza-induced IFN-γ production in vitro

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Heparinised whole blood was diluted 1:10 with serum-free RPMI 1640 medium and incubated with either A/California/07/2009 (H1N1) or A/Perth/16/2009 (H3N2) allantoic fluids (1.6 × 10⁶ PFU). Phytohemagglutinin PHA-M (Sigma-Aldrich, Dorset, UK) was used as a positive control and egg allantoic fluid was used as a negative control. Blood was stimulated for 4 days at 37 °C, after which plasma supernatants were collected and cryopreserved at −80 °C. The Ferret IFN-γ ELISA Development Kit (ALP) (Mabtech, Nacka, Sweden) was used to determine the quantity of IFN-ɣ secreted by cells in the blood as described^{18 (link)}.

Marriott A.C., Gooch K.E., Brown P.J., Ryan K.A., Jones N.J., Merredew N., Wiblin N., Dibben O., Bright H., Hallis B., Whittaker C.J, & Carroll M.W. (2021). Severity of heterosubtypic influenza virus infection in ferrets is reduced by live attenuated influenza vaccine. NPJ Vaccines, 6, 43.

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Cytokine-Induced Stat Phosphorylation in hPBMCs

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hPBMCs (3 × 10⁶ cells/tube) were incubated with a test compound for 30 min at 37 °C, and then treated with IL-2 (100 ng/mL), IL-6 (100 ng/mL), IL-23 (100 ng/mL), IFN-α (100 ng/mL), or GM-CSF (1 ng/mL) for an additional 15 min. To terminate the stimulation, the cells were fixed with Fixation Buffer (BD Biosciences) for 10 min at 37 °C. The fixed cells were incubated with Perm Buffer II (BD Biosciences) for 30 min on ice and then incubated with fluorochrome-labeled anti-CD3, anti-CD4, or anti-phospho-Stat antibodies for 30 min at room temperature. The cytokine-induced Stat phosphorylation was analyzed and quantified using a Cytomics FC500 (Beckman Coulter Inc., Brea, CA). In the case of IL-23 stimulation, hPBMCs were precultured with 10 μg/mL phytohemagglutinin (PHA)-M (Sigma-Aldrich Co.) for 3 days to enhance their response to IL-23.

Tanimoto A., Ogawa Y., Oki C., Kimoto Y., Nozawa K., Amano W., Noji S., Shiozaki M., Matsuo A., Shinozaki Y, & Matsushita M. (2014). Pharmacological properties of JTE-052: a novel potent JAK inhibitor that suppresses various inflammatory responses in vitro and in vivo. Inflammation Research, 64(1), 41-51.

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Isolation and Transduction of Anti-GD2 CAR T Cells

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PBMCs were isolated from healthy donors or GBM affected patients (as approved by the Institutional Review Board), separated by a density gradient (Lymphoprep; Alere Technologies AS, Oslo, Norway; #1114545) and then plated in RPMI 1640 (Gibco; #52400-017) with 1% FBS, 1% glutamine, and 1% penicillin–streptomycin. Nonadherent cells were collected and prestimulated for 48 h in RPMI 1640 supplemented with 10% heat-inactivated defined FBS (HyClone Laboratories, UT, USA; #SH30070.03), 500 UI/mL rhInterleukin-2 (rhIL-2, Proleukin, Clinigen Healthcare Ltd, Stafforshire DE14 2WW, UK; #801313AY), and 7 μg/mL Phytohemagglutinin (PHA-M, Sigma-Aldrich; #L8629) at the concentration of 1 × 10⁶ cells/mL. Isolated T cell population underwent retroviral transductions to express a second-generation anti-GD2 CAR; for details refer to Prapa et al.^{16 (link)}, and GFP control vector.

Prapa M., Chiavelli C., Golinelli G., Grisendi G., Bestagno M., Di Tinco R., Dall’Ora M., Neri G., Candini O., Spano C., Petrachi T., Bertoni L., Carnevale G., Pugliese G., Depenni R., Feletti A., Iaccarino C., Pavesi G, & Dominici M. (2021). GD2 CAR T cells against human glioblastoma. NPJ Precision Oncology, 5, 93.

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