Healthy donor whole blood was obtained from the City of Hope blood donor center, and HIV-donor whole blood used for HIV reactivation study was obtained from ZenBio, which HIV donors received for mobilization assay obtained from Dr. Cardoso’s Lab at City of Hope. Blood was ficolled for peripheral blood mononuclear cells (PBMCs),22 (link) and CD3+ cells were then isolated from PBMCs using the EasySep Human T Cell Isolation Kit according to manufacturer’s instructions (STEMCELL Technologies). Freshly isolated CD3+ cells were activated with Dynabeads human T-activator CD3/CD28 (Life Technologies) and transduced 24 h after with a LV encoding for either crLV- or LV-derived NIH45-46CAR:4-1BB:CD3ζ/huEGFRt at an MOI of 2 and expanded in the presence of 50 U/mL recombinant human interleukin-2 (rhIL-2; Novartis) and 0.5 ng/mL interleukin-15 (rhIL-15; Novartis), which was supplemented every 48–72 h during the course of ex vivo expansion for 14–21 days.22 (link) Mock-transduced T cells (no LV) were used as control. HIV expression was determined by an Alliance HIV-1 p24 ELISA (Perkin Elmer).
Alliance hiv 1 p24 elisa
Manufactured by PerkinElmer
Sourced in United States
The Alliance HIV-1 p24 ELISA is a laboratory test kit used for the qualitative detection of the HIV-1 p24 antigen in human serum or plasma samples. It is intended for use as an aid in the diagnosis of HIV-1 infection.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Recombinant human interleukin 2 rhil 2, by Novartis (1 mentions)
Rhil 15, by Novartis (1 mentions)
Easysep human t cell isolation kit, by STEMCELL (1 mentions)
24 well plate, by Corning (1 mentions)
Fitc mouse anti human cd4, by BD (1 mentions)
Mouse igg1 fitc, by BD (1 mentions)
Pe mouse anti human cd184, by BD (1 mentions)
Pierce ldh cytotoxicity assay kit, by Thermo Fisher Scientific (1 mentions)
Fitc mouse anti human cd3, by BD (1 mentions)
Alexa fluor 647 coupled anti gfp, by BD (1 mentions)
Pe mouse igg1, by BioLegend (1 mentions)
Pe mouse anti human cd11b mac 1, by BD (1 mentions)
Fitc mouse igg2a, by BD (1 mentions)
Mouse igg2a pe, by BD (1 mentions)
3 protocols using alliance hiv 1 p24 elisa
1
HIV Reactivation Assay Using Genetically Modified T Cells
2
HIV Infection Kinetics in CEM.CCR5+ Cells
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CAR-expressing CEM.CCR5+ (1 × 106) were infected with either X4- (LAI, NL4-3, YU2) or R5- (Bal) tropical virus and incubated for 18 h before a PBS wash. Cells were resuspended in 1 mL in a 24-well plate (Corning) and cultured for 14 days. On day 7 and 14, supernatant was collected, and HIV expression was determined by an Alliance HIV-1 p24 ELISA (Perkin Elmer, Waltham, MA, USA).
3
Antibody Detection and Cytotoxicity Assays
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The following antibodies were used: Alexa Fluor 647–coupled anti-GFP (catalog no. 565197, BD Pharmingen); Alexa Fluor 647 rat immunoglobulin G2a (IgG2a), κ isotype control (catalog no. 400526); Alexa Fluor 647 anti-human CD195 (catalog no. 313712) from BioLegend; phycoerythrin (PE) mouse IgG1, κ isotype control (catalog no. 550617); PE mouse IgG2a, κ isotype control (catalog no. 553457); fluorescein isothiocyanate (FITC) mouse IgG2a, κ isotype control (catalog no. 555573); FITC mouse IgG1 κ isotype control (catalog no. 555748); PE mouse anti-human CD184 (catalog no. 555974); FITC mouse anti-human CD4 (catalog no. 555346); FITC mouse anti-human CD3 (catalog no. 555332); and PE mouse anti-human CD11b/Mac1 (catalog no. 555388) from BD Biosciences.
The amount of lactate dehydrogenase (LDH) released into supernatants was quantified using a Pierce LDH Cytotoxicity Assay kit (catalog no. 88953, Thermo Fisher Scientific). The Alliance HIV-1 p24 ELISA (enzyme-linked immunosorbent assay) Kit (catalog no. NEK050, PerkinElmer) was used to determine the amount of capsid p24 protein in supernatants and cell lysates. LDH and p24 quantification were performed following the manufacturer’s instructions.
The amount of lactate dehydrogenase (LDH) released into supernatants was quantified using a Pierce LDH Cytotoxicity Assay kit (catalog no. 88953, Thermo Fisher Scientific). The Alliance HIV-1 p24 ELISA (enzyme-linked immunosorbent assay) Kit (catalog no. NEK050, PerkinElmer) was used to determine the amount of capsid p24 protein in supernatants and cell lysates. LDH and p24 quantification were performed following the manufacturer’s instructions.
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