Sample Prep & Detection Kits
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Accessory reagents & controlsBiochemicals
BiochemicalsProteins and Peptides
Proteins and peptidesAnti-Ly6g antibody [1A8] - mouse IgG2c (Chimeric)
Low endotoxin, Azide free.
Our first-to-market chimera with mouse IgG2c backbone, this functional antibody specifically depletes neutrophils in vivo for up to 72h.
Learn about all product ranges with our product overviews.
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Ensure that all the reagents and samples are kept on ice from steps 1–6 as this has a positive impact on cell viability.
In order to maintain sterility, perform all steps in a laminar flow hood with sterile utensils. Watch our video guide on aseptic technique.
Cut the back legs above the hip joint
The femur tends to have more marrow than the tibia.
Use only intact bones, as the bone marrow will not be sterile if they break.
Fill a petri dish with 70% ethanol.
The bones can be left in ethanol for longer while each bone is flushed, but be aware that it can soak into the bone and kill viable cells.
In a laminar flow hood using sterile utensils, cut both ends of the bone with scissors as close to the joints as possible.
Centrifuge 1–2 times in R10 media at 300 x g for 5 minutes.
Resuspend the cell pellet in R10 and count viable cells using a hemocytometer and trypan blue.
See our detailed protocol on using a hemocytometer to obtain a viable cell count.
Dilute the cells into 10 mL of R10 + 20 ng/mL GM-CSF.
Place plates in a 37°C incubator with 5% carbon dioxide.
At day 3, add an additional 10 mL of R10 + 20 ng/mL GM-CSF.
Add media very gently so as not to disturb the growing cells.
At day 6, remove half of the media (10mL).
Remove media very gently so as not to disturb the growing cells.
Briefly centrifuge the removed volume of media.
Cells can be harvested on day 8, or step 10 can be repeated to harvest cells on day 9 or 10.
Cells should be only briefly centrifuged as the DCs are only loosely adherent.
Non-adherent and loosely adherent cells in the culture in the culture supernatant can be harvested by gentle washing with PBS, and then pooled for subsequent experiments.
The majority of adherent cells will be macrophages, while those that are in suspension or loosely adherent will be dendritic cells (DCs) and F4/80+ macrophages. For a highly pure fraction, it is recommended to take measures to deplete F4/80+ cells and enrich the population for CD11c+ cells.
Most of the macrophages in suspension will dually express F4/80+ and CD11c. The F4/80+ cells can be removed via positive selection with anti-F4/80 magnetic beads. After this step, CD11c+ DCs can be positively selected from the flow-through using anti-CD11c magnetic beads.
The immature DC’s can be used immediately after purification, or further cultured if the user wishes to obtain mature cells.
DC’s are loosely adherent and can mature simply by adhering to a plate, therefore a non-tissue culture treated 96-well u-bottom plate can be used to prevent premature maturation.
Purity assessment
Suggested markers
Anti-CD11c antibody [N418] (ab33483). This is also available labeled with Biotin, FITC, APC/Cy7, PE, PE/Cy7, PerCP/Cy5.5 and as low-endotoxin, azide-free.
Anti-CD11b antibody [EPR19373] (ab184307)
Anti-F4/80 antibody [BM8] (ab16911) or Anti-F4/80 antibody [CI:A3-1] (ab6640). These antibodies are also available directly conjugated to FITC, PE or APC.