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Mouse lamina propria dissociation kit

Manufactured by Miltenyi Biotec
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The Mouse Lamina Propria Dissociation Kit is a laboratory product designed to facilitate the isolation of cells from the lamina propria of mouse intestinal tissue. It provides a standardized protocol and reagents for the efficient dissociation of the lamina propria, enabling the extraction of various cell types for downstream applications.

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Lab products found in correlation

Gentlemacs dissociator, by Miltenyi Biotec (4 mentions) Neutrophil elastase, by R&D Systems (2 mentions) Myeloperoxidase, by R&D Systems (2 mentions) Kanamycin, by Merck Group (2 mentions) Nalidixic acid, by Merck Group (2 mentions) Duoset enzyme linked immunosorbent assay, by R&D Systems (2 mentions) Interleukin il 22, by R&D Systems (2 mentions) Sybr green mix, by Quanta Biosciences (2 mentions) Qscript complementary dna cdna synthesis kit, by Quanta Biosciences (2 mentions) Ly6g antibody, by Abcam (2 mentions) Alcian blue ph 2.5 stain kit, by Vector Laboratories (2 mentions) Cl amidine hydrochloride, by Cayman Chemical (2 mentions) Il 17a, by R&D Systems (2 mentions) Rpmi 1640, by Merck Group (2 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Luria bertani broth, by Merck Group (2 mentions) Histopaque 1077 and 1119, by Merck Group (2 mentions) Serum amyloid a, by R&D Systems (2 mentions) Regulatory t cell isolation kit, by Miltenyi Biotec (1 mentions)

13 protocols using mouse lamina propria dissociation kit

1

Gut Immune Cell Isolation and Analysis

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Reagents and materials were purchased from the following sources: anti–mouse CD4 (RM4-5), CD25 (PC61) antibodies were purchased from BD Biosciences; anti-mouse IL-17A (TC11-18H10.1), IFN-γ (XMG1.2), CD90.1 (OX7) antibodies were purchased from BioLegend; anti-mouse Foxp3 (FJK-16s) and IL-22 (1H8PWSR) antibodies as well as Foxp3 staining buffer set were purchased from eBioscience; anti-mouse CD90.1 (19E.2) and anti-human CD2 (OKT11) were purchased from the hybridoma lab of Epitope Recognition and Immunoreagent Core at UAB; live/dead staining kit was purchased from Invitrogen; mouse lamina propria dissociation kit and regulatory T cell isolation kit were purchased from Miltenyi Biotec; mouse LCN-2 ELISA kit was purchased from R&D Systems; fecal DNA extraction kit was purchased from Zymo Research.
ZHAO Q., HARBOUR S.N., KOLDE R., LATORRE I.J., TUN H.M., SCHOEB T.R., TURNER H., MOON J.J., KHAFIPOUR E., XAVIER R.J., WEAVER C.T, & ELSON C.O. (2017). SELECTIVE INDUCTION OF HOMEOSTATIC TH17 CELLS IN THE MURINE INTESTINE BY CHOLERA TOXIN INTERACTING WITH THE MICROBIOTA. Journal of immunology (Baltimore, Md. : 1950), 199(1), 312-322.
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2

Isolation of Colon Lamina Propria Cells

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Lamina propria tissue of colons was dissociated to single-cell suspensions by
combining mechanical dissociation with enzymatic degradation of the
extracellular adhesion proteins using a gentleMACS dissociator and a mouse
lamina propria dissociation kit according to the manufacturer’s
instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Initially, the
intraepithelial lymphocytes (IELs) were disrupted from the mucosa by shaking the
tissue in a predigestion solution. Then, the lamina propria tissue was further
treated enzymatically and mechanically dissociated into a single-cell suspension
containing lamina propria mononuclear cells (LPMCs) by using a gentleMACS
dissociator. The resultant cell suspensions containing the LPMCs and IELs were
further purified from contaminating epithelium, stroma and dead cells by
centrifugation (1,000 × G,
45 minutes, 20 °C) on a 40/80% Percoll
gradient (Sigma-Aldrich, St. Louis, MO).
Däbritz J., Judd L.M., Chalinor H.V., Menheniott T.R, & Giraud A.S. (2016). Altered gp130 signalling ameliorates experimental colitis via myeloid cell-specific STAT3 activation and myeloid-derived suppressor cells. Scientific Reports, 6, 20584.
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3

Extraction and Quantification of Murine Immune Factors

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Histopaque-1077 and −1119, RPMI 1640, Luria-Bertani (LB) broth, bovine serum albumin (BSA), paraformaldehyde, nalidixic acid and kanamycin were procured from Sigma (St. Louis, MO). DNase I (dornase alfa, Pulmozyme®) was kindly gifted from Genentech. Duoset enzyme-linked immunosorbent assay (ELISA) kits for mouse Lipocalin2 (Lcn2), keratinocyte-derived chemokine (KC), serum amyloid A (SAA), neutrophil elastase (NE), myeloperoxidase (MPO), interleukin (IL)-22 and IL-17A were obtained from R&D Systems (Minneapolis, MN). SYBR Green mix and the qScript complementary DNA (cDNA) synthesis kit were procured from Quanta BioSciences (Gaithersburg, MD). Ly6G antibody was purchased from Abcam (Cambridge, MA). All other fine chemicals used in the present study were reagent grade and procured from Sigma. Alcian Blue (pH 2.5) Stain Kit was obtained from Vector Laboratories (Burlingame, CA). Mouse Lamina Propria Dissociation Kit was purchased from Miltenyi Biotec (Auburn, CA). Cl-amidine (hydrochloride) was obtained from Cayman chemicals (Ann Arbor, MI).
Saha P., Yeoh B.S., Xiao X., Golonka R.M., Singh V., Wang Y, & Vijay-Kumar M. (2019). PAD4-dependent NETs generation are indispensable for intestinal clearance of Citrobacter rodentium. Mucosal immunology, 12(3), 761-771.
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4

Isolation and Characterization of Intestinal Immune Cells

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Immune cells were isolated from terminal small intestinal tissue sections using mouse Lamina Propria Dissociation kit (Miltenyi) according to the manufacturer’s instructions. Following isolation, immune cells were washed with PBS and resuspended in PBS+1%BSA, and stored at 4°C before proceeding with flow cytometry staining. For flow cytometry staining, ~1X106 cells were placed in a 96-well plate and Fc receptor blocking as done using mouse Fc Block (BD biosciences) for 15 min at 4°C. Cells were then stained with an appropriate Ab mixture for surface staining at 4°C for 30 mins. Cells were washed after staining with staining buffer and fixed with fixation buffer (Biolegend). Cells were analyzed on BD LSR II (BD biosciences) and flow cytometry data was analyzed using FlowJo software (version 10.8.0; Tree star, Ashland, OR).
Jalodia R., Kolli U., Braniff R.G., Tao J., Abu Y.F., Chupikova I., Moidunny S., Ramakrishnan S, & Roy S. (2022). Morphine mediated neutrophil infiltration in intestinal tissue play essential role in histological damage and microbial dysbiosis. Gut Microbes, 14(1), 2143225.
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5

Intestinal Lamina Propria Dissociation

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Longitudinally sectioned pieces of the small intestine or the colon were
processed using a gentleMACS Dissociator and mouse lamina propria dissociation
kit (both Miltenyi Biotec), according to the manufacturer's protocol.
Dithiothreitol was excluded from the entire process. We sectioned the small
intestine into the proximal third (duodenum), the middle third (jejunum) and the
distal third (ileum).
Koyama M., Mukhopadhyay P., Schuster I.S., Henden A.S., Hülsdünker J., Varelias A., Vetizou M., Kuns R.D., Robb R.J., Zhang P., Blazar B.R., Thomas R., Begun J., Waddell N., Trinchieri G., Zeiser R., Clouston A.D., Degli-Esposti M.A, & Hill G.R. (2019). MHC class II antigen presentation by the intestinal epithelium initiates graft-versus-host disease and is influenced by the microbiota. Immunity, 51(5), 885-898.e7.
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6

Colonic Immune Cell Isolation

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Colonic tissues from controls and CR-infected WT and Pad4−/− mice were dissociated to single-cell suspensions by combining mechanical dissociation and enzymatic degradation, followed by lamina propria immune cells isolation according to manufacturer’s instructions (mouse Lamina Propria Dissociation Kit, Miltenyi Biotec).
Saha P., Yeoh B.S., Xiao X., Golonka R.M., Singh V., Wang Y, & Vijay-Kumar M. (2019). PAD4-dependent NETs generation are indispensable for intestinal clearance of Citrobacter rodentium. Mucosal immunology, 12(3), 761-771.
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7

Murine Lymphoid Tissue Dissociation

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Cell suspensions were obtained from the spleen, the PP, the MLN and the AA LN (lumbar and renal lymph nodes). Jejunum and colon sections from PBS and LCWE-injected mice were harvested and dissociated into single-cell suspensions with a gentleMACS™ Octo Dissociator (Miltenyi Biotec) and a mouse Lamina Propria Dissociation kit (Miltenyi Biotec) following the complete protocol from the manufacturer. The following antibodies against the respective murine antigens were used: IgA (mA-6E1,Thermo Fisher Scientific), CD19 (eBio1D3, Thermo Fisher Scientific), CD4 (RM4–5, Tonbo Biosciences), CD3 (145–2C11, BioLegend and Tonbo Biosciences), CD45.1 (A20, Thermo Fisher Scientific), CD45.2 (104, BioLegend), CD95 (SA367H8, BioLegend), GL-7 (GL-7, BioLegend). Dead cells were routinely excluded based on the staining of Fixable Viability dye (FVD) eFluor 506 (Thermo Fisher Scientific). Cell numbers were calculated by flow cytometry with the CountBright Absolute Counting Beads (Thermo Fisher Scientific). Stained cells were analyzed on a LSRII (BD Biosciences) and the data were processed using Flowjo (Tree Star Inc.).
Rivas M.N., Wakita D., Franklin M.K., Carvalho T.T., Abolhesn A., Gomez A.C., Chen S., Lehman T.J., Sato K., Shibuya A., Fasano A., Kiyono H., Abe M., Tatsumoto N., Yamashita M., Crother T.R., Shimada K, & Arditi M. (2019). Intestinal permeability and IgA provoke immune vasculitis linked to cardiovascular inflammation. Immunity, 51(3), 508-521.e6.
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8

Colonic Immune Cell Isolation

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Colonic tissues from controls and CR-infected WT and Pad4−/− mice were dissociated to single-cell suspensions by combining mechanical dissociation and enzymatic degradation, followed by lamina propria immune cells isolation according to manufacturer’s instructions (mouse Lamina Propria Dissociation Kit, Miltenyi Biotec).
Saha P., Yeoh B.S., Xiao X., Golonka R.M., Singh V., Wang Y, & Vijay-Kumar M. (2019). PAD4-dependent NETs generation are indispensable for intestinal clearance of Citrobacter rodentium. Mucosal immunology, 12(3), 761-771.
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9

Dissociation of Mouse Small Intestine

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The whole small intestine obtained from host mice 14 days after aHSCT was processed using a gentleMACS Dissociator (Miltenyi Biotec) and mouse lamina propria dissociation kit (Miltenyi Biotec), according to the manufacturer's protocol.
Tsubokura Y., Yoshimura H., Satake A., Nasa Y., Tsuji R., Ito T, & Nomura S. (2022). Early administration of lenalidomide after allogeneic hematopoietic stem cell transplantation suppresses graft‐versus‐host disease by inhibiting T‐cell migration to the gastrointestinal tract. Immunity, Inflammation and Disease, 10(9), e688.
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10

Extraction and Quantification of Murine Immune Factors

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Histopaque-1077 and −1119, RPMI 1640, Luria-Bertani (LB) broth, bovine serum albumin (BSA), paraformaldehyde, nalidixic acid and kanamycin were procured from Sigma (St. Louis, MO). DNase I (dornase alfa, Pulmozyme®) was kindly gifted from Genentech. Duoset enzyme-linked immunosorbent assay (ELISA) kits for mouse Lipocalin2 (Lcn2), keratinocyte-derived chemokine (KC), serum amyloid A (SAA), neutrophil elastase (NE), myeloperoxidase (MPO), interleukin (IL)-22 and IL-17A were obtained from R&D Systems (Minneapolis, MN). SYBR Green mix and the qScript complementary DNA (cDNA) synthesis kit were procured from Quanta BioSciences (Gaithersburg, MD). Ly6G antibody was purchased from Abcam (Cambridge, MA). All other fine chemicals used in the present study were reagent grade and procured from Sigma. Alcian Blue (pH 2.5) Stain Kit was obtained from Vector Laboratories (Burlingame, CA). Mouse Lamina Propria Dissociation Kit was purchased from Miltenyi Biotec (Auburn, CA). Cl-amidine (hydrochloride) was obtained from Cayman chemicals (Ann Arbor, MI).
Saha P., Yeoh B.S., Xiao X., Golonka R.M., Singh V., Wang Y, & Vijay-Kumar M. (2019). PAD4-dependent NETs generation are indispensable for intestinal clearance of Citrobacter rodentium. Mucosal immunology, 12(3), 761-771.
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