iPSCs were first differentiated into hematopoietic progenitor cells following manufacturer’s instructions using a commercially available kit (StemCell Technologies #05310) as described before (Andreone et al., 2020 (link)). HPCs positive for identity markers CD34, CD43, and CD45 were transferred to a plate containing primary human astrocytes and co-cultured using media C adapted from a previous study (Pandya et al., 2017 (link)). Once floating cells in co-culture are predominantly (>80%) mature microglia, cells were plated for 3–4 days prior to experiments. Full characterization of human iPSC-derived microglia and additional details on the differentiation protocol has been published elsewhere (Andreone et al., 2020 (link)). All the experiments using GRN+/+ and GRN−/− iMG were performed in IMDM (Gibco) media supplemented with 10% defined FBS (Gibco), 1% Penicillin/Streptomycin (Gibco), 20 ng/mL of hIL3 (Peprotech), 20 ng/mL of hGM-CSF (Peprotech) and 20 ng/mL of hM-CSF (Peprotech) (referred to as “C+++ Media”). Immunostainings of PGRN were conducted using the anti-progranulin goat polyclonal antiserum (R&D Systems # AF2420, 1:250) to verify the absence of immunoreactivity in knockout iMG.
Af2420
Manufactured by R&D Systems
AF2420 is a protein that functions as a cytokine and is involved in the regulation of immune responses. It is used in research applications to study the biological activities of this protein.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using af2420
1
iPSC-Derived Microglial Characterization
2
Quantification of Progranulin Levels in Tissue Lysates
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Levels in tissues were measured after homogenization in RIPA buffer and quantification with an ELISA assay (developed in-house) in tissue lysate supernatants using human progranulin capture antibody mix for plate coating (R&D Systems, AF2420, at 1:100), and mouse anti-human progranulin detection antibody (R&D Systems, MAB2420, at 1:1,000). To normalize between samples, total protein levels were measured using the Pierce BCA protein assay. The final progranulin values reported are ng mg−1 protein (ng ml−1 progranulin in lysate divided by mg ml−1 total protein concentration). All samples were measured in triplicate. For technical replicate measurements, if at least one replicate measurement was below the lower limit of quantitation (LLOQ), the sample is reported as below quantitative limit. For graphing and statistical purposes, values below the LLOQ (below quantitative limit) were calculated as 0 ng mg−1. Data were acquired by microplate reader, Varioskan LUX, and analyzed using SkanIt RE 5.0.1 software (Thermo Fisher Scientific).
3
Validating PGRN and PSAP Antibody Specificity
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To validate the specificity of the PGRN goat antibody (R&D Systems #AF2420), antibody was incubated overnight at 4 °C with recombinant human PGRN protein (R&D Systems #2420-PG, amino acids 18–593) in a mass ratio of 1:200. Similarly, the PSAP rabbit antibody (R&D Systems #AF8520) was incubated with recombinant human PSAP protein (Sino-Biologicals, Beijing, China, #16224-H08H) in the same ratio. Control and protein-absorbed antibodies were diluted to the optimal concentrations for immunohistochemistry and reacted with sections using the above-described enzyme immunohistochemistry procedure. PGRN-absorbed antibody prepared in the same manner was also used for western blots.
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