Blood samples were drawn by venipuncture into sodium-heparin vacutainers. Lymphocyte cell culturing was carried out for the collected blood samples on the same day.[11 (link)] 0.5 mL of the whole blood sample was added to 5.0 mL of RPMI 1640 culture medium [Hi Media]. Subsequently, 1.2 mL of Foetal Bovine Serum (FBS) [Hi Media] and 0.3 mL of Phytohaemagglutinin [GIBCO] were also added to the cultures. Incubation of the cultures was carried out at 37°C for a period of 72 h.
Phytohaemagglutinin
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Phytohaemagglutinin is a lectin protein derived from the red kidney bean (Phaseolus vulgaris). It is used in cell culture and biomedical research applications to induce mitogenic activation and proliferation of T lymphocytes.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 culture medium, by HiMedia (1 mentions)
Foetal bovine serum (fbs), by HiMedia (1 mentions)
Ferrous sulphate, by Merck Group (1 mentions)
Ethyl acetate, by BD (1 mentions)
Potassium persulfate, by Merck Group (1 mentions)
Brain heart infusion broth, by Thermo Fisher Scientific (1 mentions)
Multiskan ex plate reader, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Ferric chloride, by Merck Group (1 mentions)
Blood agar, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bhi agar, by Thermo Fisher Scientific (1 mentions)
Cytochalasin b, by Thermo Fisher Scientific (1 mentions)
2 2 azinobis 3 ethylbenzothiazolin 6 sulfonic acid, by Merck Group (1 mentions)
Uv 1800 uv vis spectrophotometer, by Shimadzu (1 mentions)
1 1 diphenyl 2 picrylhydrazyl, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
8 protocols using phytohaemagglutinin
1
Lymphocyte Cell Culture from Blood
2
Antioxidant Assay Protocol
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Ethyl acetate and H2O2 were purchased from BDH, UK. dimethyl sulfoxide (DMSO), 1`1` diphenyl 2 picrylhydrazyl (DPPH), ferric chloride, ferrous sulphate, methanol, 2,4,6-triphyridyl-s-triazine (TPTZ), and 2,2-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid (ABTS), potassium persulfate, ascorbic acid, gallic acid, trolox and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-etrazoliumbromide (MTT) were purchased from Sigma-Aldrich Inc. (USA). Dulbecco’s modified eagle’s medium (DMEM), Rosswell Park Memorial Institute (RPMI) 1640 medium, foetal bovine serum (FBS), penicillin/streptomycin, glutamine, phytohaemagglutinin and cytochalasin B were purchased from Gibco BRL (USA). Bleomycin (BLM) was purchased from United Biotech, India. Brain heart infusion (BHI) broth, BHI agar, blood agar and anaerobic sachets were from Oxoid (UK). Defibrinated sheep blood was from the Veterinary Research Institute, Gannoruwa, Sri Lanka. UV-1800 UV-Vis spectrophotometer, Shimadzu, Japan and Multiskan Ex plate reader from Thermoscientific, USA, were used for antioxidant and MTT assays respectively.
3
Stimulation and Analysis of MAIT Cells
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Ninety‐six‐well round bottom plates (Sigma‐Aldrich, Castle Hill, NSW, Australia) were coated with 50 μL of 5 μg mL−1 anti‐CD3 (HIT3a; BD Pharmingen, San Jose, CA, USA) in PBS at 37°C for 4 h prior to stimulation experiments. Purified MAIT cells were cultured in TexMacs (Miltenyi Biotec) supplemented with 2% (v/v) fetal bovine serum (FBS) (Sigma‐Aldrich) and 1× Penicillin‐Streptomycin (Sigma‐Aldrich) in the presence of IL‐2 (50 U mL−1), IL‐7 (10 U mL−1), Phytohaemagglutinin) (1:1000; Gibco, Grand Island, NY, USA) and CD28 (1 μg mL−1, clone CD28.2; BD Pharmingen). This media is herein referred to as “stimulation media.”
For time course experiments, approximately 100 000 purified MAIT cells were cultured in 100 μL of stimulation media, with 35 μL of supernatant removed at each time point and replaced with 35 μL of fresh stimulation media. For flow cytometry analysis, MAIT cells were usually stimulated as above, with the addition of 10 ng mL−1 phorbol 12‐myristate 13‐acetate (Sigma, Castle Hill, NSW, Australia) and 1 μg mL−1 ionomycin (Sigma, Castle Hill, NSW, Australia) in the presence of GolgiPlug (1:1000; BD Biosciences) for the final 5 h prior to harvesting.
For time course experiments, approximately 100 000 purified MAIT cells were cultured in 100 μL of stimulation media, with 35 μL of supernatant removed at each time point and replaced with 35 μL of fresh stimulation media. For flow cytometry analysis, MAIT cells were usually stimulated as above, with the addition of 10 ng mL−1 phorbol 12‐myristate 13‐acetate (Sigma, Castle Hill, NSW, Australia) and 1 μg mL−1 ionomycin (Sigma, Castle Hill, NSW, Australia) in the presence of GolgiPlug (1:1000; BD Biosciences) for the final 5 h prior to harvesting.
4
Healthy Volunteer Blood Lymphocyte Culture
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Samples of venous blood were collected from four healthy, non-smoking volunteers, two women and two men, aged between 20 and 25 years, with no history of pesticide exposure. The donors were informed about the study and signed the consent form allowing the use of their blood samples. The investigation observed the ethical standards laid down by the Declaration of Helsinki.
Blood lymphocytes were cultured at 37 °C for 70 h by adding 0.3 mL of whole blood to 4.7 mL of Ham's F-10 medium (Sigma) supplemented with 20 % foetal bovine serum (Sigma), 2 % phytohaemagglutinin (Gibco, Paisley, UK), and antibiotics (penicillin at the concentration of 100 IU mL -1 and streptomycin at 100 µg mL -1 ) (IE Ulagay, Istanbul, Turkey.)
Blood lymphocytes were cultured at 37 °C for 70 h by adding 0.3 mL of whole blood to 4.7 mL of Ham's F-10 medium (Sigma) supplemented with 20 % foetal bovine serum (Sigma), 2 % phytohaemagglutinin (Gibco, Paisley, UK), and antibiotics (penicillin at the concentration of 100 IU mL -1 and streptomycin at 100 µg mL -1 ) (IE Ulagay, Istanbul, Turkey.)
5
Coculture of aNAV B cells and CD4+ T cells
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aNAV B cells and CD4+ T cells were sorted using FACSAria III (BD Biosciences). Sorted cells were cocultured in 96-well plates precoated with anti-human CD3 mAbs (BioLegend, San Diego, California, USA), in the presence of 1 µg/mL R848, 50 ng/mL IL-2, 10 µg/mL calf thymus DNA at 37°C, 5% CO2 for 5 days. Cells were harvested and assessed for T-cell proliferation and effector Th-cell polarisation by carboxyfluorescein succinimidyl ester (CFSE) proliferation assay and intracellular cytokine staining, respectively. Controls included cultures of CD4+ T cells with anti-CD3 mAbs, R848, IL-2 and calf thymus DNA. Positive controls of T-cell proliferation were stimulated with 2% (v/v) phytohaemagglutinin (Thermo Fisher Scientific, Massachusetts, USA).
For T-cell proliferation assay, CD4+T cells were labelled with 10 µM CFSE (BioLegend) before coculture. On day 5, cells were harvested and stained with Zombie Red dye, then CD4+ T-cell proliferation was determined by flow cytometry.
For T-cell proliferation assay, CD4+T cells were labelled with 10 µM CFSE (BioLegend) before coculture. On day 5, cells were harvested and stained with Zombie Red dye, then CD4+ T-cell proliferation was determined by flow cytometry.
6
Metaphase Chromosome Spread Preparation
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Metaphase chromosome spreads were prepared from whole blood cell cultures, following the protocol described in [25 (link)]. Briefly, a small amount (approx. 40 μL) of peripheral blood was cultured for a week at 30 °C in Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich, St. Louis, MO, USA), enriched with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), 0.5% penicillin/streptomycin solution (Thermo Fisher Scientific, Waltham, MA, USA), 1% l -glutamine (Sigma-Aldrich, St. Louis, MO, USA), 3% phytohaemagglutinin (Thermo Fisher Scientific, Waltham, MA, USA) and 1% lipopolysaccharide (Sigma-Aldrich, St. Louis, MO, USA). Chromosome preparations were made following standard procedures including a colcemid treatment for 3.5 h, hypotonization with 0.563% KCl for 30 min and fixation in 3:1 methanol: acetic acid. Chromosomal preparations from all specimens were stained with conventional Giemsa solution. C-banding was performed as described by [26 (link)] with slight modifications, i.e., the slides were aged at 65 °C for 1 h, soaked in 0.2N HCl for 20 min, then in 5% Ba(OH)2 solution for 4.5 min at 45 °C and then rinsed in 0.2N HCl. Finally, the slides were soaked in 2× saline-sodium citrate (SSC) buffer for 1 h at 60 °C, rinsed in distilled water, and stained with 4′,6-diamidino-2-phenylindole (DAPI).
7
Culturing blood for metaphase cells
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Blood cultures were set up by inoculating 1 mL of freshly drawn blood into 75 mL tissue-culture flasks containing 9 mL of peripheral blood (PB)-Max medium composed of Roswell Park Memorial Institute (RPMI) 1640 medium with 15.0% fetal bovine serum (FBS), 1.0% penicillinstreptomycin and 3.0% of phytohaemagglutinin (Gibco-Invitrogen Ltd., Paisley, Ren-frewshire, UK). To collect metaphase cells, cultures were exposed to 100 μL of 10 μg/mL colcemid (Gibco-Invitrogen Ltd.) 2 hours prior to cell harvesting [23 (link),24 (link)].
8
LPS and PHA Protocol for Cell Stimulation
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Lipopolysaccharide from Escherichia coli serotype 0127:B8 was obtained from Sigma (St Louis, MO, USA); phytohaemagglutinin was from Invitrogen (PHA, Invitrogen, Paisley, UK). All reagents were purchased at the highest purity available.
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