Plasma samples containing high levels of F8 were heat inactivated at 58°C for 90 min. A standard curve was generated by serial dilution of the commercial human F8 (Refacto, Pfizer) in F8‐deficient plasma. Serial dilutions of all samples were done in F8‐deficient plasma and then mixed 1:1 with 100% Refacto (Pfizer) diluted in F8‐deficient plasma. All experimental samples and controls were then incubated at 37°C for 2 h. After that, all samples and the standard curve were analysed by aPTT following the manufacturer’s manual.
Refacto
Manufactured by Pfizer
Refacto is a laboratory equipment product manufactured by Pfizer. It is designed to assist in the handling and processing of various samples and materials in a research or laboratory setting. The core function of Refacto is to provide a reliable and efficient tool for researchers and laboratory professionals to conduct their work.
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Lab products found in correlation
6 protocols using refacto
1
Quantifying Factor VIII Activity
2
Conditioning Newborn and Adult HA Mice
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Animal studies were approved by the Animal Care and Use Committee of the Università del Piemonte Orientale "A. Avogadro" (Novara, Italy) and the by the Italian Ministry of Health with the authorization no. 758/2021-PR, and the Ethical Review Board of the Universidad Pablo de Olavide (Seville, Spain) according to the Eurpoean Union regulations. In vivo experiments were performed on recipient newborn and adult HA mice in a C57BL/6 background (C57BL/6-HA).18 (link) Donor FL and BM cells were isolated from green fluorescent protein-positive (GFP+) mice in a C57BL/6 background (C57BL/6-Tg(ACTbEGFP)1Osb/J, strain #:003291).18 (link) FL cells were obtained from embryonic day (E)11 or E13 of gestation.23 (link),24 (link) Timed breedings of GFP+ transgenic mice were established to obtain the fetuses. Vaginal plugs were checked daily and the day a plug was detected was considered as E0. In order to generate recipient conditioned newborn mice, HA pregnant females were treated with BU (15.5 mg/kg; Sigma-Aldrich) plus 1 international unit (IU) recombinant human (rh) FVIII (Re-Facto®, Pfizer) for 48 hours (h) and 24 h (BU group) or 24 h (1/2 BU1x group) before delivery, while adult mice (8 weeks old) received BU injections (30 mg/kg/injection) 48 h and 24 h before transplantation.
3
Comparative Evaluation of Moroctocog Alfa
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The test product was moroctocog alfa (AF‐CC), manufactured using the albumin‐free process (ReFacto AF, Pfizer, Collegeville, PA). The reference product was moroctocog alfa (ReFacto, Pfizer) manufactured using the process containing albumin.2 Each subject received single doses of 50 IU/kg as 2‐min infusions of each of the study medications in a randomized fashion. The dose was calculated on the basis of the subject's actual body weight as measured at the screening visit and on the labeled potency of the study drug. After completion of the first study period and a minimum washout of 5 days, subjects received the alternate treatment.
4
Evaluating Factor VIII Activity in Plasma Samples
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Briefly, nine parts of blood were collected by retro‐orbital withdrawal into one part of buffered trisodium citrate 0.109 M (5T31.363048; BD, Franklin Lakes, NJ, USA). Blood plasma was collected after samples centrifugation at 3,000 rpm at 4°C for 15 min.
To evaluate F8 activity, chromogenic assay was performed on plasma samples using a Coatest® SP4 FVIII‐kit (K824094; Chromogenix, Werfen, Milan, Italy) according to the manufacturer’s instructions. Standard curve was generated by serial dilution of commercial human F8 (Refacto, Pfizer). Results are expressed as International Units (IU) per decilitre (dl).
Activated partial thromboplastin time (aPTT) was measured on plasma samples with Coatron M4 (Teco, Bünde, Germany) using the aPTT programme following the manufacturer’s manual.
To quantify F8 antigen levels, the ELISA kit (FVIII‐AG; VisuLize FVIII ELISA kit, Affinity Biologicals, Arcore, Italy) was used according to the manufacturer’s instructions.
To evaluate F8 activity, chromogenic assay was performed on plasma samples using a Coatest® SP4 FVIII‐kit (K824094; Chromogenix, Werfen, Milan, Italy) according to the manufacturer’s instructions. Standard curve was generated by serial dilution of commercial human F8 (Refacto, Pfizer). Results are expressed as International Units (IU) per decilitre (dl).
Activated partial thromboplastin time (aPTT) was measured on plasma samples with Coatron M4 (Teco, Bünde, Germany) using the aPTT programme following the manufacturer’s manual.
To quantify F8 antigen levels, the ELISA kit (FVIII‐AG; VisuLize FVIII ELISA kit, Affinity Biologicals, Arcore, Italy) was used according to the manufacturer’s instructions.
5
Evaluating BDD-FVIII and mRNA Therapies
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Mice were 8- to 12-week old F8 exon 16 knockout C57BL/6 mice (a kind gift from Prof H.H. Kazazian, Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA). Mice were injected intravenously with recombinant BDD-FVIII (rFVIII, Refacto®, Pfizer, 150 IU/kg), or with mRNA (1 to 5 μg) formulated in TransIT® (100 to 350 μL final volume). Blood was collected from the retro-orbital sinus 6, 24, 48, 72 or 120 h following the injection of mRNA. Plasma was kept frozen at −80°C until use. Animals were handled in agreement with local ethical authorities (approval by Charles Darwin ethics committee, authorization #3335 2015121718044892). FVIII:Ag, FVIII:C, anti-FVIII IgG and FVIII inhibitors were measured as described in the Online Supplementary Methods.
6
FVIII-Induced Splenocyte Proliferation
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Total NHP splenocytes were plated in flat-bottom 96-well plate (3 × 105 cells/well) in X-vivo-15 (Lonza). Cells were left unstimulated or stimulated with increasing dose of FVIII.BDD protein (ReFACTO, Pfizer) in triplicates. Anti-NHP CD3 (clone CD3-1) stimulation served as positive control as polyclonal inducer of T-cell proliferation. After 5 days of culture, 1µCi/well of 3H-Thymidine was added and incubated for additional 16 h. Cell proliferation was indirectly quantified by measuring 3H-Thymidine incorporation. The stimulation index (SI) was obtained as the ratio between the mean counts per minutes (cpm) in each stimulated condition and the mean counts of the unstimulated cell.
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