Single cell suspensions were generated by pressing spleens through a cell strainer (70 μm) and naïve B cells were purified using a CD43-depletion kit (Miltenyi, 130-049-801). Cells were lysed in 4% SDS containing phosphatase and protease inhibitors (Merck, 4906845001 and 05892970001). Protein concentration was measured by BCA assay (Thermo Fisher, 23225) and concentration was adjusted to 800 μg/ml before the addition of Laemmli buffer. 20 μl per sample were loaded onto 10% polyacrylamide gels and blotted onto a PVDF membrane. Membranes were blocked (5% BSA in TBST) and stained overnight with primary antibody (pp65, Cell Signaling, 3033; pIRAK4, Abnova, MAB2538; pBTK, Cell Signaling, 5082, GAPDH, Cell Signaling, 5174). Membranes were washed and incubated with HRP-coupled secondary antibody for one hour at room temperature. After washing, membranes were incubated with ECL solution (Amersham) and imaged on a ChemiDoc (Bio-Rad). Densitometric analysis was performed using ImageJ.
Cd43 depletion kit
Manufactured by Miltenyi Biotec
The CD43-depletion kit is a laboratory tool designed to deplete CD43-positive cells from biological samples. CD43 is a surface marker expressed on various cell types, including T cells, B cells, and monocytes. The kit provides a method to selectively remove these CD43-positive cells, allowing for the isolation and study of the remaining cell populations.
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Lab products found in correlation
Bca assay, by Thermo Fisher Scientific (1 mentions)
Ecl solution, by Cytiva (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
P btk, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Lsr fortessa cytometer, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
2 protocols using cd43 depletion kit
1
Splenic Naive B Cell Protein Analysis
2
Lymphoid Organ Cell Isolation and Analysis
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Lymphoid organ cells [spleen, bone marrow, mesenteric lymph nodes (mLN), inguinal lymph nodes (iLN), or Peyer's patches (PPs)] were isolated and stained as previously described (27 (link)). Briefly, single cell suspensions were stained with appropriate antibodies (Supplementary Table 1 ) in PBS supplemented with 2% BSA and 2 mM EDTA for cell surface staining. Intracellular staining was performed using the Foxp3/Transcription factor staining buffer set (eBioscience) according to the provider recommendation. Flow cytometry analyses were performed on a BD LSR Fortessa cytometer and cell sorting experiments for qPCR analysis were performed using a BD FACS AriaII cell sorter. Data were analyzed with the Flowjo software (TreeStar, Ashland, OR). Splenic B cell magnetic enrichments for in vitro differentiation assay were performed using the CD43 depletion kit (Miltenyi Biotec) according to the manufacturer's recommendations.
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