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Multiscreen ip plate

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MultiScreen-IP plates are a line of lab equipment designed for efficient cell culture and assay applications. They feature a porous membrane that allows for the separation and filtration of cell samples. The plates are available in various well formats to accommodate different experimental needs.

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Lab products found in correlation

Immunospot reader, by Cellular Technology (4 mentions) Alkaline phosphatase conjugated streptavidin, by Jackson ImmunoResearch (3 mentions) Bcip nbt substrate kit, by Bio-Rad (3 mentions) An 18, by Thermo Fisher Scientific (2 mentions) Alkaline phosphatase conjugated goat anti mouse igm, by Southern Biotech (2 mentions) Tween 20, by Merck Group (2 mentions) Concanavalin a (cona), by Merck Group (2 mentions) Bcip nbt plus substrate, by Mabtech (2 mentions) Streptavidin alkaline phosphatase, by Thermo Fisher Scientific (2 mentions) Immunospot imaging analyzer system, by Cellular Technology (2 mentions) Immunospot analyzer, by Cellular Technology (2 mentions) Alkaline phosphatase conjugate substrate kit, by Bio-Rad (2 mentions) Streptavidin alkaline phosphatase, by Abcam (1 mentions) Anti mouse igm antibody, by Southern Biotech (1 mentions) Mouse igg1 isotype control clone mopc 21, by BioXCell (1 mentions) Ctl immunospot reader, by Cellular Technology (1 mentions) Ra 6a2, by Thermo Fisher Scientific (1 mentions) Hrp conjugated streptavidin, by Southern Biotech (1 mentions) Bcip nbt, by Merck Group (1 mentions) Goat anti mouse ig, by Southern Biotech (1 mentions)

26 protocols using multiscreen ip plate

1

Enzyme-based IgM Secreting Cell Assay

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BM cells were added to sterile MultiScreen IP-Plates (Millipore, MSIPS4510) according to manufacturer’s protocol after coating wells with unlabelled anti-mouse IgM antibody (Southern Biotech). IgM producing cells were visualized using streptavidin alkaline phosphatase (Abcam) followed with BCIP/NBT. Each spot represented an IgM secreting cell.
Rosenfeld S.M., Perry H.M., Gonen A., Prohaska T.A., Srikakulapu P., Grewal S., Das D., McSkimming C., Taylor A.M., Tsimikas S., Bender T.P., Witztum J.L, & McNamara C.A. (2015). B-1b Cells Secrete Atheroprotective IgM and Attenuate Atherosclerosis. Circulation research, 117(3), e28-e39.
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2

IFN-γ ELISPOT Assay for Mycobacterial Antigens

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IFN-γ Elispot assay was performed as previously described [24 (link)], with some modifications. Briefly, multiscreen-IP plates (Millipore, Bedford, MA) were coated with anti-IFN-γ mAb (An-18, eBioscience) at 5μg/ml in PBS. BMDCs were pre-pulsed with Mtb fractions or recombinant protein antigens overnight. In a blocking assay, Mtb antigen-pulsed BMDCs were pre-incubated with mouse IgG or anti-Qa-2 mAb (20-8-4) [48 (link)] for 30 min before the assay. Enriched CD8+ T cells from infected mice (5×103−2×104) were mixed with BMDCs stimulator cells (5×104/well) in RPMI 10 medium and plated in triplicate wells. After 18h incubation at 37°C, plates were washed using PBS-Tween (PBS and 0.05% Tween 20) and incubated for 2h at room temperature with biotinylated anti-IFN-γ mAb (R4.6A2, eBioscience). Plates were then washed and incubated with streptavidin-conjugated alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA). After 1 h incubation at room temperature, plates were developed with a BCIP/NBT substrate kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Spots were counted using an ImmunoSpot reader (Cellular Technology, Shaker Heights, OH).
Shang S., Siddiqui S., Bian Y., Zhao J, & Wang C.R. (2016). Nonclassical MHC Ib-restricted CD8+ T Cells Recognize Mycobacterium tuberculosis-Derived Protein Antigens and Contribute to Protection Against Infection. PLoS Pathogens, 12(6), e1005688.
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3

ELISPOT Assay for Naive B Cell Enumeration

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ELISPOT assay was carried out as previously described (36 (link)). In brief, sort-purified, naive B cells were distributed onto MultiScreen*-IP Plates (Millipore) pre-coated with goat anti-mouse Ig (H+L) and then incubated in RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin for 4 h at 37°C and 5% CO2. Plates were treated with alkaline phosphatase-conjugated goat anti-mouse IgM (Southern Biotechnology Associates) and developed with 5-bromo-4-chloro-3-indolyl phosphate/p-NBT chloride substrate (KPL). IgM-secreting B cells were enumerated using Phoretix Expression software (NonLinear Dynamics).
Tsuji N., Rothstein T.L, & Holodick N.E. (2020). Antigen Receptor Specificity and Cell Location Influence the Diversification and Selection of the B-1a Cell Pool with Age. Journal of immunology (Baltimore, Md. : 1950), 205(3), 741-759.
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4

IFN-γ ELISpot Assay for CD8+ T Cell Response

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IFN-γ ELISpot assay was performed as previously described [11 (link)], with some modifications. Briefly, multiscreen-IP plates (Millipore, Bedford, MA) were coated with anti-IFN-γ mAb (AN-18, BioLegend, San Diego, CA) at 5μg/ml in PBS. Enriched CD8+ T cells from infected mice were incubated with MHC II-/- BMDCs with media alone or 5μM of Mtb peptide, in duplicate. To confirm Qa-1 restriction, BMDCs were pre-incubated with 2 μg/ml anti-Qa-1 blocking mAb (6A8.6F10.1A6, BD, Franklin Lakes, NJ) or mouse IgG1 isotype control (clone MOPC-21) (BioXCell, West Lebanon, NH) prior to adding peptide and lymphocytes. After 18h incubation at 37°C, plates were washed using PBS/0.05% Tween 20 and developed using biotinylated α-IFN-γ mAb (R4.6A2, eBioscience, San Diego, CA), followed by streptavidin-conjugated alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA) and a BCIP/NBT substrate kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Spots were counted using an ImmunoSpot reader (Cellular Technology, Shaker Heights, OH).
Bian Y., Shang S., Siddiqui S., Zhao J., Joosten S.A., Ottenhoff T.H., Cantor H, & Wang C.R. (2017). MHC Ib molecule Qa-1 presents Mycobacterium tuberculosis peptide antigens to CD8+ T cells and contributes to protection against infection. PLoS Pathogens, 13(5), e1006384.
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5

IFN-γ ELISPOT Assay for Mouse T Cells

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B6 and B6.E+ splenic CD4+ T cells were obtained by incubating cells with anti-CD4 microbeads for 30 min. at 4°C and flowing samples through magnetized LS positive selection columns (Miltenyi Biotec). 1.5 × 106 T cells were cultured for 48 h with equal numbers of irradiated Balb/c or SJL splenocytes, 20ng/mL phorbol 12-myristate 13-acetate (PMA) + 1μg/mL ionomycin, or medium only in 96-well MultiScreen-IP plates (Millipore) coated with purified anti-IFNγ (RA-6A2; eBioscience). Plates were then incubated with biotinylated anti-IFNγ (XMG1.2; eBioscience) and streptavidin-conjugated HRP (Southern Biotech), developed with 5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium (BCIP/NBT; Sigma-Aldrich), read on a CTL ImmunoSpot reader, and analyzed using CTL ImmunoSpot 4.0 (Cellular Technology Limited).
Ni P.P., Wang Y, & Allen P.M. (2014). Both Positive and Negative Effects on Immune Responses by Expression of a Second Class II MHC Molecule. Molecular immunology, 62(1), 199-208.
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6

Cytokine Profiling of Liver and Spleen

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Enzyme-linked immunosorbant assay (ELISA) was performed to detect IFN-γ or IL-17A cytokine secretion. Cytometric bead assay (CBA) was performed to detect IL-4, IL-6, IL-10, IL-13, IL-23, TNF-α, and GM-CSF cytokine secretion. Single cell suspensions of liver or spleen were incubated with α-GalCer (200 ng/ml) or heat-killed SA (HKSA) (106 CFU) for 48 h, then supernatant was collected for ELISA or CBA. For ELISPOT assay, Multiscreen-IP plates (Millipore) were coated with 10 µg/ml mouse anti-IFN-γ or anti-IL-17A overnight in PBS, then blocked with cRPMI. Total liver lymphocytes or T cell enriched liver lymphocytes were cultured with total SA lipid or SA lipid fraction-pulsed BMDCs for 18-20 h before assay development, detailed assay protocol published previously (33 (link)). Developed ELISPOT plates were imaged using an ImmunoSpot reader (Cellular Technology Ltd.).
Genardi S., Visvabharathy L., Cao L., Morgun E., Cui Y., Qi C., Chen Y.H., Gapin L., Berdyshev E, & Wang C.R. (2020). Type II Natural Killer T Cells Contribute to Protection Against Systemic Methicillin-Resistant Staphylococcus aureus Infection. Frontiers in Immunology, 11, 610010.
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7

Amyloid Beta ELISPOT Assay

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MultiScreen-IP plates (Millipore, Billerica, MA) were washed with 70% ethanol, rinsed three times with phosphate-buffered saline (PBS), and then coated with 100 μl of Aβ42 (4 μg/ml) in PBS at 4°C overnight. Plates were washed six times with PBS and blocked with RPMI with 2% BSA for 2 h at room temperature. Rested splenocytes and bone marrow cells were plated in 200 μl of medium and incubated 48 h at 37°C. Plates were washed six times in PBS with 0.25% Tween 20 (Sigma) and incubated with 100 μl of 1:250 HRP conjugated goat-anti-rabbit IgG or goat-anti-rabbit IgA for 1 h at room temperature. The plates were again washed with PBS-T and a final wash in PBS, and developed with 100 μl of AEC substrate for 20 to 30 min at room temperature, and the reaction was stopped with deionized water. Spots were air dried and automatically counted with an ELISPOT reader.
Lambracht-Washington D., Fu M., Wight-Carter M., Riegel M, & Rosenberg R.N. (2017). Evaluation of a DNA Aβ42 Vaccine in Aged NZW Rabbits: Antibody Kinetics and Immune Profile after Intradermal Immunization with Full-Length DNA Aβ42 Trimer. Journal of Alzheimer's Disease, 57(1), 97-112.
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8

Evaluating SUDV and EBOV Peptide-Specific T-cell Responses

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Multiscreen-IP plates (Millipore) were activated, coated and blocked according to the kit manufacturer’s instructions (Mabtech). 2 × 105 freshly isolated splenocytes were added per well in triplicates with either 2 µg/ml the LYDRLASTV peptide present in both SUDV and EBOV GP38 (link), or the RPHTPQFLF peptide derived from SUDV GP38 (link), medium alone or Concanavalin A (Sigma-Aldrich). After 20 ± 2 h of incubation at 37 °C with 5% CO2, plates were developed as recommended by the manufacturer (Mabtech). Plates were analyzed using the Immunospot analyzer and software (Immunospot).
Öhlund P., García-Arriaza J., Zusinaite E., Szurgot I., Männik A., Kraus A., Ustav M., Merits A., Esteban M., Liljeström P, & Ljungberg K. (2018). DNA-launched RNA replicon vaccines induce potent anti-Ebolavirus immune responses that can be further improved by a recombinant MVA boost. Scientific Reports, 8, 12459.
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9

Quantification of Antibody-Secreting Cells

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Milipore multiscreen-IP plates (Milipore, MA) were prepared per manufacturer’s instruction and coated with appropriate antibodies in PBS: 2ug/mL Goat Anti-Mouse Ig (Southern Biotech), or 4–8ug/mL anti-human IgG (Rockland Biosciences, NY). The following day, plates were washed and blocked with complete medium. Bulk cell suspensions were prepared, diluted appropriately and incubated in complete IMDM (10% FCS, 1.0% Pen/Strep) for 18 hours at 37°C and 5% CO2. Standard ELISPOT procedures were followed using biotinylated anti-mouse IgG (Sigma) or anti-human IgG (mABtech) followed by streptavidin-ALP (mABTech). Assay was developed with NBT/BCIP Substrate (ThermoScientific) and analyzed using CTL Immunospot 5.0 software (Cellular Tech Ltd). Data were calculated as antibody-secreting (ASC) spots/106 lymphocytes.
Tedesco D., Thapa M., Gumber S., Elrod E.J., Rahman K., Ibegbu C.C., Magliocca J.F., Adams A.B., Anania F, & Grakoui A. (2016). CD4+ Foxp3+ T cells promote aberrant IgG production and maintain CD8+ T cell suppression during chronic liver disease. Hepatology (Baltimore, Md.), 65(2), 661-677.
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10

ELISPOT Assay for B Cell Enumeration

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ELISPOT assay was carried out as previously described (38 (link), 39 (link)). In brief, indicated numbers of sort-purified B1a cells and PCs or 5×104 splenocytes were distributed onto MultiScreen-IP Plates (Millipore) pre-coated with goat anti-mouse IgG+M+A (H+L) cross absorbed antibodies (Bethyl Lab) and then incubated in RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, 100 U/ml penicillin, and 100 µg/ml streptomycin for 4h at 37°C and 5% CO2. Plates were treated with F(ab’)2 fragment, goat anti-mouse IgG or anti-mouse IgM, HRP-conjugated, (Jackson ImmunoResearch), or goat anti-mouse IgG2c, HRP conjugated, (Bethyl Lab) and developed with AEC (2-amino-9-ethylcarbzole) substrate (Sigma-Aldrich). IgM-, IgG- and IgG2c-secreting B cells were enumerated using ImmunoSpot analyzer/software (Cellular Technology Limited).
Wu Y.Y., Georg I., Díaz-Barreiro A., Varela N., Lauwerys B., Kumar R., Bagavant H., Castillo-Martín M., Salem F.E., Marañón C, & Alarcón-Riquelme M.E. (2015). Concordance of Increased B1 Cell Subset and Lupus Phenotypes in Mouse and Human Dependent on BLK Expression Levels. Journal of immunology (Baltimore, Md. : 1950), 194(12), 5692-5702.
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