Color development buffer

Manufactured by Bio-Rad

The Color Development Buffer is a laboratory solution used to facilitate the development of color in various biochemical assays and immunodetection techniques. It serves as a supporting medium to optimize the color-producing reaction, allowing for the visualization and quantification of target analytes.

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Lab products found in correlation

Multiscreen ip 96 well plates, by Merck Group (3 mentions) Streptavidin alp, by Mabtech (3 mentions) Clone 7 b6 1, by Mabtech (2 mentions) Anti ifn γ antibody clone 1 d1k, by Mabtech (1 mentions)

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Development buffer (8 citations)

3 protocols using color development buffer

Granzyme B ELISPOT Assay for HIV Antigens

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Mabtech Granzyme B (clone GB10, catalog 3486-2 A) ELISPOT assays against whole gene product peptides with overlap 15-mer coverage of HIV-Gag (catalog ARP-12425), HIV-Env (catalog ARP-12540), HIV-Pol (catalog ARP-12438), and HIV-Nef (catalog ARP-12545) from the NIH HIV Reagent Program and CMVpp65 peptide pool (catalog PM-PP65-2) were performed in triplicate. Multiscreen IP 96-well plates (MilliporeSigma) were coated with 7.5 μg/mL of anti–Granzyme B antibody (clone GB10, Mabtech catalog 3486-2 A) in sterile water and incubated overnight. Plates were washed, and PBMCs were added at 100,000–200,000 cells/well and stimulated with peptide pools and 0.5% DMSO and phytohemagglutinin at 2 μg/mL as negative and positive controls, respectively. Plates were incubated overnight; washed and biotinylated antibody was added (anti–Granzyme B antibody clone MT8610 from Mabtech) and incubated for 2 hours. Plates were developed with Streptavidin-ALP from Mabtech and with Color Development Buffer (Bio-Rad). Plates were washed and dried overnight and spots were counted. Responses against whole gene product peptide pools were background subtracted (thus, nonzero responses were more than 1 time background), but no other ad hoc empirical cutoff was applied — consistent with other studies examining correlations with objectively reported T cell responses as assessed by ELISPOT assay.

Copertino DC J.r., Holmberg C.S., Weiler J., Ward A.R., Howard J.N., Levinger C., Pang A.P., Corley M.J., Dündar F., Zumbo P., Betel D., Gandhi R.T., McMahon D.K., Bosch R.J., Linden N., Macatangay B.J., Cyktor J.C., Eron J.J., Mellors J.W., Kovacs C., Benko E., Bosque A, & Jones R.B. (2023). The latency-reversing agent HODHBt synergizes with IL-15 to enhance cytotoxic function of HIV-specific T cells. JCI Insight, 8(18), e169028.

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HIV Interferon-Gamma ELISPOT Assay Protocol

Cited 1 time

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HIV interferon-gamma ELISPOT assays were performed as previously described and reported on samples from the last on-ART time point.^{9 (link)}In brief, Multiscreen IP 96-well plates (Millipore) were coated with 0.5 ug/mL of anti-interferon-gamma antibody (clone 1-D1K, MAbtech) in phosphate-buffered saline and incubated overnight. Plates were washed, peripheral blood mononuclear cells were added at 2×10⁵cells per well and HIV peptide pools (10 ug/ml/peptide) and phytohemaggultinin (2ug/mL) were added. Plates were incubated overnight, washed and secondary antibody was added (clone 7-B6–1, Mabtech) and incubated for 1 hour. Plates were developed with Streptavidin-ALP (MAbtech) and developed with Color Development buffer (Bio-Rad). Plates were washed, dried overnight and spots were counted.

Keating S.M., Jones R.B., Lalama C.M., Bosch R.J., McMahon D., Hampton D., Cyktor J., Eron J.J., Mellors J.W., Busch M.P, & Gandhi R.T. (2019). HIV Antibodies Decline During Antiretroviral Therapy but Remain Correlated with HIV DNA and HIV-specific T Cell Responses. Journal of acquired immune deficiency syndromes (1999), 81(5), 594-599.

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IFN-γ ELISPOT Assay for HIV and CMV

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IFN-γ enzyme-linked immune absorbent spot (ELISPOT) assays against HIV-gene product peptide pools and a CMV-pp65 peptide pool were performed as previously described [18 (link)]. In brief, Multiscreen IP 96-well plates (Millipore) were coated with 0.5 μg/mL of anti-IFN-γ antibody (clone 1-D1K, Mabtech, Sweden) in phosphate-buffered saline and incubated overnight. Plates were washed, PBMCs were added at 2 × 10⁵ cells per well, and HIV peptide pools or CMV-pp65 peptide pool (10 μg/mL/peptide) and phytohemagglutinin (2 μg/mL) were added. Plates were incubated overnight, washed and secondary antibody was added (clone 7-B6–1, Mabtech) and incubated for 1 hour. Plates were developed with Streptavidin-ALP (Mabtech) and Color Development Buffer (Bio-Rad, Hercules, CA). ELISPOT responses were background subtracted prior to data analysis.

Ward A.R., Thomas A.S., Stevenson E.M., Huang S.H., Keating S.M., Gandhi R.T., McMahon D.K., Bosch R.J., Macatangay B.J., Cyktor J.C., Eron J.J., Mellors J.W, & Jones R.B. (2022). No evidence that circulating HIV-specific immune responses contribute to persistent inflammation and immune activation in persons on long-term ART. AIDS (London, England), 36(12), 1617-1628.

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