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Member since: Mar 03, 2005
From: North Carolina, United States
Status: Cell & Tissue Culture, Antibody Technologies Moderator
My points: 575    what's this
Name: Sambhudho Mukherjee

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Oct 6, 2010    10:54 AM

Replied to topic in Cell Culture and Tissue Culture forum   HEK 293 cells (Flp In Trex system)

293s adhere better and 'flatten' out in the presence of FBS. If you do a dose response (0, 0.25, .5, 1 and 5% FBS), you'll clearly see this phenomenon.

Oct 5, 2010    11:34 AM

Replied to topic in Antibody Based Technologies forum   Background DAB Precipitate

Also, ensure that your PBS is free of Fe/Ca/Mg - or just use DI 18 mho water for the DAB.

Oct 5, 2010    11:33 AM

Replied to topic in Antibody Based Technologies forum   Background DAB Precipitate

How critical is the Ni - i.e. are you okay with brown (and a nuclear stain like methyl green) or do you need the black staining? For the former - eliminate Ni. If you need black immunostaining, use a new stock of H2O2 and Ni when you make your DAB mix. I usually use the Vectastain system with the ...

Sep 28, 2010    12:08 PM

Replied to topic in Cell Culture and Tissue Culture forum   cell viability assays

MTT is a rather crude end-point assay to measure redox potential (and indirectly viability: live cells have a membrane potential and mitochondrial potential, high metabolic states result in mitochondrial biogenesis/enhanced activation, as does enhanced cell proliferation). MTT has a small dynamic r ...

Sep 27, 2010    04:29 PM

Replied to topic in Cell Culture and Tissue Culture forum   Giant brown cells among the lymphocytes

Are these in round or flat bottom plates? Can you do a quick DAPI stain in situ when it happens, and observe in an inverted fl scope? It might actually be T cell 'activation clusters' (T cells can function as weak +ve and moderate -ve (tolerogenic) APCs in T-T interactions) especially if they are i ...

Sep 22, 2010    09:50 AM

Replied to topic in Antibody Based Technologies forum   What is the best fitting curve for ELISA standard Curve ?

<<Rajeshwari patel: In my oppiniun, Non- liner fit is suitable for ELISA analysis.>> Actually, thats a terrible idea for most ELISAs, and if you are doing that, you need to read up and revise! e.g. When you set up your standards as serial double dilutions, you expect halving absorb ...

Sep 20, 2010    03:17 PM

Replied to topic in Cell Culture and Tissue Culture forum   PBMC isolation from frozen whole blood

gDNA isolation - yes; virus culture - almost certainly no; flow cytometry/cytospin analysis: maybe.

Sep 18, 2010    02:11 PM

Replied to topic in Cell Culture and Tissue Culture forum   Cryopreservation of T lymphocytes

We use 5%DMSO, 5%Gibco opt and 90% HI serum as freezing medium for later functional analysis. Note that the DMSO does cause some amount of cell permeability, and the thawed cells are really fragile. Bottom line: be very gentle during Ab/tetramer staining/washes. AB serum should work just fine - try ...

Sep 9, 2010    11:27 AM

Replied to topic in Antibody Based Technologies forum   recombinant monoclonal antibody

Glycosylation and marker variability are magnified in non-reducing native conditions - a more stringent technique will show ~155-165 kD for IgG isotypes. 2x55kD+2x25kD in reducing SDS gels.

Sep 8, 2010    10:24 AM

Replied to topic in Antibody Based Technologies forum   Buying antibodies. Opinions?

For monoclonals, I usually just look at the published clone(s), and pick up the cheapest supplier with the right color/fluorophore - usually this is BDPharmingen or eBioscience or BioLegend in the first tier, and CalTag/Invitrogen, Abcam and Abnova or Novus in the second. For polyclonals and many i ...

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