Cells were cultured in RPMI 1640 culture medium supplemented with 10% foetal bovine serum (FBS), 2 mM L-glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin (Invitrogen). CD3 stimulation was carried out using plate-bound functional grade anti-human CD3 antibody (eBioscience). Five μg/ml antibody diluted in PBS was added to each culture well before incubating at 4°C for 24 h. The wells were washed with PBS to remove any unbound antibody, before 2 μg/ml functional grade anti-human CD28 antibody (eBioscience) was added to the culture medium to provide appropriate co-stimulation. PHA-P stimulation was carried out by adding 10 μg/ml PHA-P (Sigma-Aldrich) directly to the culture medium.
Pha p
Manufactured by Merck Group
Sourced in United States, Belgium, Germany
PHA-P is a laboratory instrument designed for the measurement and analysis of physiological parameters. It is capable of recording and processing data related to various biological functions.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Hil 2, by Thermo Fisher Scientific (3 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (3 mentions)
Blasticidin, by InvivoGen (2 mentions)
Puromycin, by InvivoGen (2 mentions)
Lgm 3 lymphocyte growth medium, by Lonza (2 mentions)
Tlrl picw, by InvivoGen (2 mentions)
Percoll, by GE Healthcare (2 mentions)
Geneticin, by Thermo Fisher Scientific (2 mentions)
Cytochalasin b, by Merck Group (2 mentions)
Ja 17 rotor, by Beckman Coulter (2 mentions)
Peg 1500, by Merck Group (2 mentions)
Vysis lsi 21 spectrumorange probe, by Abbott (2 mentions)
Live dead fixable aqua dead cell kit, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa, by BD (2 mentions)
3h thymidine, by PerkinElmer (2 mentions)
Interleukin 2 (il 2), by Merck Group (2 mentions)
62 protocols using pha p
1
T Cell Activation with CD3/CD28 and PHA
2
Generation of Stable Knockdown and Overexpression Jurkat and T Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For generating pools of shRNA knockdown Jurkat and CEMx174 lines, cells were plated at 106 cells/mL in RPMI-FBS complete and transduced with 107 RT units of viral vector per 106 cells, followed by selection with 1 μg/ml puromycin (InvivoGen, cat# ant-pr-1). To generate stable gag-gfp or gfp expressing Jurkat cells, cells were transduced as for shRNA KD above, followed by selection with 5 μg/mL blasticidin (InvivoGen, cat# ant-bl-1) at day 3 after transduction.
CD4+ T cells were stimulated in RPMI-FBS complete, with 50 U/ml IL-2 and 5 μg/mL PHA-P (Sigma, cat# L-1668). After 3 days, T cells were washed and replated at 3 × 106 cells/mL in RPMI-FBS complete, with 50 U/ml IL-2. Cells were transduced with 108 RT units of viral vector per 106 cells followed by selection in 2 μg/mL puromycin.. After selection, cells were re-plated in RPMI-FBS complete with 50 U/ml IL-2 at 3 × 106 cells/mL in RPMI-FBS complete and transduced again with the indicated GFP vectors, 108 RT units of viral vector per 106 cells. Transduced T cells were analyzed two weeks after the 2nd transduction.
CD4+ T cells were stimulated in RPMI-FBS complete, with 50 U/ml IL-2 and 5 μg/mL PHA-P (Sigma, cat# L-1668). After 3 days, T cells were washed and replated at 3 × 106 cells/mL in RPMI-FBS complete, with 50 U/ml IL-2. Cells were transduced with 108 RT units of viral vector per 106 cells followed by selection in 2 μg/mL puromycin.. After selection, cells were re-plated in RPMI-FBS complete with 50 U/ml IL-2 at 3 × 106 cells/mL in RPMI-FBS complete and transduced again with the indicated GFP vectors, 108 RT units of viral vector per 106 cells. Transduced T cells were analyzed two weeks after the 2nd transduction.
3
Rhesus Macaque T Cell Immune Response Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The T cell immune responses in rhesus macaques were detected using PBMCs with commercially available Monkey IFN-γ and IL-2 ELISpot assay kits (Mabtech) and an Monkey IL-4 ELISpot assay kit (U-CyTech). The cryopreserved rhesus macaque PBMCs were thawed and cultured with prewarmed AIM-V medium. For the IFN-γ, IL-2 and IL-4 ELISpot assays, 1.0×105 PBMCs were stimulated with a final concentration of 1 μg/ml for each RBD peptide (Table S5 ). The test for each rhesus macaque was performed in two or three technical repetitions. Dimethyl sulfoxide (DMSO) served as an unstimulating control, and phytohemagglutinin (PHA-P, Sigma) and CELL STIMULATION COCKTAIL (Thermo Fisher) were used as positive controls. After 24 h of stimulation with RBD peptide pools, the streptavidin-HRP substrate (for IFN-γ and IL-2) or AEC substrate (IL-4) was added to the plate. The spots were counted by Beijing Dakewei Biotechnology Co., Ltd. The results are background (DMSO treated group) subtracted and normalized to SFC/106 PBMCs.
4
PBMC Isolation, Stimulation, and HIV-1 Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMC) from healthy donors were isolated and stimulated for 3 days with 1 mg/mL of phytohemagglutinin‐P (PHA‐P, Sigma) and 5 IU/mL of recombinant human interleukin‐2 (rHuIL‐2, Roche). PBMC were growth under CO2 in a humid atmosphere at 37 °C in RPMI‐1640 GlutaMAX medium supplemented with antibiotics (penicillin, streptomycin, neomycin), 10 % fetal calf serum (FCS, previously inactivated by heat) and 10 UI/mL of rHuIL‐2. The HIV‐1 LAI strain was previously described.[19] Resistant viral strains are from the NIH AIDS Research & Reference Reagent Program (HIV‐1–52534‐2, HIV‐1–56252‐1, HIV‐1–71361‐1, and HIV‐1–7324‐1) carry mutations in RT.[16]
5
Coculture of Salivary Epithelial Cells and PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human peripheral blood mononuclear cells (PBMCs) were purchased from Lonza. Healthy human salivary gland epithelial cells (SGECs) were isolated and cultured as we reported previously [24 (link)]. The SGEC-PBMC coculture experiment was based on a published protocol [25 (link)]. Briefly, SGECs were seeded at 1?×?105 cells per well into 12-well plates and cultured in Keratinocyte serum-free medium (SFM, Life technology) with poly I:C (5?μg/ml, InvivoGen, tlrl-picw) for 12 hours to allow attachment, stimulation of autoantigen synthesis, and IL7 expression essential for SS progression. After removing the SFM and washing with PBS, 2?×?104 PBMCs per well were added in LGM-3 lymphocyte growth medium (Lonza) containing 10% FBS and phytohemagglutinin-P (PHA-P, 5?μg/ml, Sigma-Aldrich, L8754) to activate T cells. After 4 days of coculture, SGECs and PBMCs were harvested together and analyzed for gene expression by qRT-PCR.
6
CFSE-labeled T-cell Proliferation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
5 × 105 of the T-lymphocytes isolated from 5 healthy donors were labelled with 1 μM of Carboxyfluorescein succinimidyl ester (CFSE) (BD Pharmingen) at 37°C for 20 min, then added to a 24-well tissue culture plate in the presence of phytohaemagglutinin (PHA-P, 5 mg/ml) (Sigma-Aldrich) and co-cultured with neutrophils (N) obtained from 8 HL or 5 healthy subjects (matched for sex and age) at ratio 1:2 and 1:8. The T-cell proliferation measured by CFSE dilution was evaluated by flow cytometry after 72 hours. The experiments were conducted in parallel in presence of 200 μM nor-NOHA.
7
Microcell-mediated chromosome transfer in RPE1 cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MMCT was performed as previously described37 (link) with modifications. A9 cells were cultured to ~70% confluence, and treated with 75 ng/ml colcemid for 48 hours. Cells were collected and resuspended in 1:1 DMEM: Percoll (GE Healthcare Biosciences) with 10 µg/ml Cytochalasin B (Sigma-Aldrich), and spun at 17,000 rpm for 75 minutes in a Beckman JA17 rotor. Supernatant was collected and filtered through 10 and 5 µm filters. Approximately 2×106 RPE1 cells were collected and mixed with filtered microcells, treated with 100 µg/ml PHA-P (Sigma-Aldrich) for 30 minutes, and fused by PEG 1500 (Sigma-Aldrich) in solution. Hybrid cells were plated and cultured for 48 hours, and selected with 500 µg/ml Geneticin (Life Technologies) for 12–14 days. Standard G-band analysis was performed at Karyologic, Inc. SNP array was performed at the DFCI microarray core, using the Human Mapping 250k-Nsp platform. Fluorescent in situ hybridization was performed with the Vysis LSI 21 SpectrumOrange probe (Abbott Molecular) according to the manufacturer’s instructions.
8
HIV-1 Infection Assay in Activated PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors’ buffy coats (Red Cross of Luxembourg, Luxembourg, Luxembourg) using Ficoll-Hypaque gradient as indicated previously (Sigma-Aldrich, Liège, Belgium). PBMCs were stimulated using 10 μg/mL phytohemagglutinin (PHA-P, Sigma Aldrich) for 48 h and recombinant IL-2 (10 U/mL, Roche, Sigma-Aldrich, Liège, Belgium) for another 24 h. Stimulated PBMCs were infected by the HIV-1 reference strains IIIB/ADA-M or primary clinical isolates expanded in culture from anonymized left-over samples (Centre Hospitalier de Luxembourg, Luxembourg, Luxembourg) in the presence or absence of drugs replaced every other day during 7 days. P24 production was measured in supernatants by ELISA (Perkin Elmer, Brussels, Belgium). Efavirenz (EFV) and azidothymidine (AZT) were obtained from Sigma_Aldrich. Enfuvirtide (T20) was purchased from Eurogentec (Seraing, Belgium).
9
PBMC Isolation, Activation, and HIV-1 Infection
10
PBMC Propagation and Inactivation of JR-FL Virus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Replicating JR-FL virus was propagated from a stock provided by the NIH AIDS Reagent Repository (CAT#395, donated by Dr Irvin Chen), by cell-free infection of uninfected human peripheral blood mononucleocytes (PBMCs) activated in RPMI medium containing 20% FBS, 50μg/ml gentamycin, 5μg/ml PHA-P (Sigma Cat# L1668) and 5% IL-2 for 12–24 hours followed by washing and resuspension in RPMI supplemented with only 20% FBS and 5% IL-2 for infection. Virus supernatant was inactivated using 1mM aldrithiol and then was concentrated in the same manner as VLPs for use in gel analyses.
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?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!