To determine DT40 cells doubling time, cultures of 5 × 104 cells pr ml per condition were set and counted in duplicate by haemocytometer every 6 h for 4 days. Data were analysed with GraphPad Prism and doubling time was determined by nonlinear regression of data to Exponential (Malthusian) growth equation (Extended Data Fig. 8b ). Immunoglobulin gene conversion was monitored by the conversion of IgM− cells to IgM+ cells by flow cytometry. Fluctuation analysis was performed as described22 (link) with modifications. In brief, 4 single-cell clones of DT40 WT or Fam72a−/− were stained with anti-IgM-PE (1:40, clone M-1, SouthernBiotech) and 12 populations of 5 × 104 IgM− cells were sorted for each clone. The proportion of IgM-gain was measured by flow cytometry using the same staining after two weeks of expansion (same time of expansion) and five days later for the Fam72a−/− cells (to achieve the same number of divisions as WT at two weeks). Data were analysed with FlowJo and GraphPad Prism.
Anti igm pe
Manufactured by Southern Biotech
Anti-IgM-PE is a fluorescently labeled antibody that binds to the IgM immunoglobulin. It can be used for the detection and analysis of IgM-expressing cells in various applications, such as flow cytometry and immunofluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria, by BD (1 mentions)
Pfuturbo, by Agilent Technologies (1 mentions)
Gl 183, by Beckman Coulter (1 mentions)
Fes172, by Beckman Coulter (1 mentions)
Pei transfection reagent, by Polyplus Transfection (1 mentions)
Anti kir2ds4, by Beckman Coulter (1 mentions)
Anti igg pe, by Southern Biotech (1 mentions)
Facscalibur, by BD (1 mentions)
Cellquest, by BD (1 mentions)
Anti mouse cd16 cd32 mouse fc block, by BD (1 mentions)
Anti cd5 apc, by BD (1 mentions)
Flojo software, by Tree Star (1 mentions)
Anti cd21 apc, by BD (1 mentions)
Anti cd23 fitc, by BD (1 mentions)
Anti igd pe, by BD (1 mentions)
Anti cd4 pe, by BD (1 mentions)
Anti cd3 fitc, by Santa Cruz Biotechnology (1 mentions)
First strand superscript 3 buffer, by Thermo Fisher Scientific (1 mentions)
Dtt and h2o, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
5 protocols using anti igm pe
1
Measuring DT40 Cell Doubling Time and Immunoglobulin Gene Conversion
2
Immunoglobulin Switch and Mutation Analysis
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Aid-/- splenic B cells were activated to switch to IgG3 and infected after one day with unmutated or mutated AID-pMIG or empty vector, pMIG. Four independent cultures for each retroviral (RV) infection were set up, and 2 days after infection, cultures were split and fed with fresh medium and switch inducers. Viable cells were isolated by flotation on Lympholyte 3 and 4 days after infection, and GFP+IgM+ cells from each culture were sorted independently on a FACSAria (Becton Dickson) after staining with anti-IgM-PE (Southern Biotech) and 7AAD. Two experiments were performed for each RV-AID analyzed, except for ΔAID analyzed on days 3 and 4, and A192K and F198S analyzed on day 3, where one experiment was performed for each. PfuTurbo (Agilent) (error rate = 1.3 x 10−6) was used to amplify a 748 bp 5’ GL Sμ fragment. Primers were: 5u3 (forward primer)5’-AATGGATACCTCAGTGGTTTTTAATGGTGGGTTTA-3’ [52 (link)] and m2R (reverse primer) 5’-GCTACTCCAGAGTATCTCATTTCAGATC-3’ [45 (link)]. Significance of difference of mutation frequency from FL-AID was determined by a chi-square test.
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Aid-/- splenic B cells were activated to switch to IgG3 and infected after one day with unmutated or mutated AID-pMIG or empty vector, pMIG. Four independent cultures for each retroviral (RV) infection were set up, and 2 days after infection, cultures were split and fed with fresh medium and switch inducers. Viable cells were isolated by flotation on Lympholyte 3 and 4 days after infection, and GFP+IgM+ cells from each culture were sorted independently on a FACSAria (Becton Dickson) after staining with anti-IgM-PE (Southern Biotech) and 7AAD. Two experiments were performed for each RV-AID analyzed, except for ΔAID analyzed on days 3 and 4, and A192K and F198S analyzed on day 3, where one experiment was performed for each. PfuTurbo (Agilent) (error rate = 1.3 x 10−6) was used to amplify a 748 bp 5’ GL Sμ fragment. Primers were: 5u3 (forward primer)
3
Transient Transfection of HEK-293T Cells with KIR Alleles
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HEK‐293T cells were transiently transfected using the linear polyethylenimine derivative jetPEI transfection reagent (Polyplus, New York, NY) following the manufacturer's instruction. The pcDNA 3.1 plasmids used in the study code for: 2DL1*002, *003, *004, *012; 2DL2*001; 2DL3*002, *005; 2DS1*001, *002; 2DS2*001, 2DS4*001, and 2DS5*002. Forty‐eight hours after the transfection, the cells were tested with a panel of anti‐KIR2D mAb followed by anti‐IgG‐PE or anti‐IgM‐PE (Southern Biotechnology) by immunofluorescence and flow cytometric analysis (FACSCalibur and Cell Quest software, BD Biosciences). The mAbs used in these experiments were: 143211 (R&D Systems, Minneapolis, MN), 11PB6 (anti‐KIR2DL1/S1 and anti‐KIR2DL3 allotypes carrying E35 and R50, Miltenyi Biotec, Bergisch Gladbach, Germany), HP‐MA4 (anti‐KIR2DL1/S1/S3/S5, Biolegend, San Diego, CA), GL‐183 (anti‐KIR2DL2/L3/S2, Beckman Coulter), and FES172 (anti‐KIR2DS4, Beckman Coulter).
4
Immune Cell Phenotyping by Flow Cytometry
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Splenocytes, thymocytes, and peritoneal cavity cells were resuspended in PBS containing 1% FBS and Rat Anti-Mouse CD16/CD32 Mouse BD Fc Block (BD Biosciences). The following antibody combinations were used: anti-CD4-PE (BD Biosciences, clone GK1.5) and anti-CD8a-PerCP (BD Biosciences, clone 53–6.7); anti-IgD-PE (BD Biosciences, clone 11-26c.2a) and anti-IgM-APC (SouthernBiotech, clone 1B4B1); anti-CD5-APC (BD Biosciences, clone 53–7.3) and anti-IgM-PE (SouthernBiotech, clone 1B4B1); and anti-CD21-APC (BD Biosciences, clone 7G6) and anti-CD23-FITC (BD Biosciences, clone B3B4). Flow cytometry was performed with a BD FACSCaliber and Flojo software (Treestar).
5
Purification and Characterization of RVFV-Specific B Cells
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PBMCs were purified from rabbit 8315 seven days after the third boost using a Lymphoprep (STEMCELL Technology) density gradient. PBMCs were cryopreserved in FBS plus 10% DMSO. Fluorescence-activated cell sorting of cryopreserved PBMCs was performed. PBMCs were stained with anti-CD3-FITC (Santa Cruz Biotechnology), anti-IgM-PE (Southern Biotech), anti-IgG-PerCP-Cy5.5 (Santa Cruz Biotechnology) and hexahistidine-tagged RVFV-Gn. Cells were washed and anti-HIS-APC (Abcam) was added. CD3-IgM-IgG+RVFV Gn+ cells were sorted into individual wells containing RNase OUT (Invitrogen), First Strand SuperScript III buffer, DTT and H2O (Invitrogen) and RNA was converted into cDNA (SuperScript III Reverse Transcriptase, Invitrogen) using random hexamers following the manufacturer’s protocol.
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