How to avoid ulcers in s.c. injected tumors ?

Recently, I implanted breast MDA-MB-231 tumor cells in NSG mice. Surprisingly, some mice showed ulceration in their tumors at tumor size around 200-300 mm3 much below the humane endpoint. I never observed such problem with other tumor xenografts I did in the past such as small cell lung cancer H146 and ALL MOLT-4 xenografts. Although I used CB-17 SCID mice for those studies. I am wondering whether this is common with this cell line or mice strain or the combination of both? Any suggestions or personal experience will be appreciated.

Thanks

We have run across a tumor cell line with a high-incidence of ulceration formation (leading to early euthanization in tumor-growth experiments). During a conversation with another group the idea of using Matrigel during injections was suggested as a means of preventing this.

Does anyone have any relevant experience with this solution (Subcutaneous injections)?

Greetings, good people,

I kindly request assistance in converting concentrations from µg/ml to µM.

While calculating the concentrations to evaluate compounds that may inhibit cathepsin S (the enzyme of interest) and assess their cytotoxicity using the MTT assay, I used units in "µg/ml." Now, I intend to convert them to µM, as most previous studies have utilized this unit.

The compounds in question are chlorogenic acid, quercetin, and procyanidin. According to the literature, the conversion is given by the formula: µM = µg/ml ÷ molecular weight (kD).

Regarding the conversion of g/mol to kD, we said 1 kD (kiloDalton) is equivalent to 1,000 g/mol.

For chlorogenic acid, with a molecular weight of 354.31 g/mol. Is it correct to say 10 µg/ml is equivalent to 28.22 µM?

This comes from this calculation.

µM = µg/ml ÷ (molecular weight in g/mol ÷ 1,000)

= 10 µg/ml ÷ (354.31 g/mol ÷ 1,000)

= 28.22 µM

I have 10-colour panel with following fluorochromes PerCPCy5-5, FITC, BV785, BV605, BV421, DAPI, APC-Cy7, Alexa Fluor 700, APC, PE-Cy7. In single colour controls everything is compensated correctly,  but in full stained panel I see that APC vs APC-Cy7 is like uncompensated. When I used FMO for BV785 this issue is disappeared. Has anyone observed this issue? Do you have an explanation for it?    

Dear all, 

I have tried to use 40:80% Percoll to isolate tumor-infiltrating lymphocytes from tumor tissue of CT26 BALB/c mice model. But when I used this method after collagenase digestion and centrifugation (300rpm, 25 min), no separated parts of any cell types were found. The tumor cells were still mixed in 40% Percoll layer which it should truly be sedimented at the bottom of the tube. I have tried to used the other concentrations of Percoll (75%, 70% and 65%) instead of 80% but it showed the same results. Does anyone have suggestions or experiences about this? I will very appreciate it.

Thanks!!       

We don't manage to subculture those cells in our lab. After thawing, we didn't observed any growth and their viability decreased slowly until death.

conditions of culture are:

- Incubator: 37,5°C, 5% Co2

- medium: Expi expression medium (provided by the supplier), pH measured = 7,5

- Cells were cultured in sterilized and cell culture treated erlenmeyer with vent caps. And they were on an orbital agitator at 110 RPM.

Thank you very much if you can provide some tips/informations about those cells.

Hi,

I am using Absolute counting beads to count the number of neutrophils in mouse peripheral blood. I was wondering if somebody has done similar work. I am using Count Bright beads from ThermoFisher.

i am collecting 50ul of blood ( collected in EDTA coated tubes), staining with 1A8 and CD45 antibody for 15-20 min and then using ammonium chloride lysis buffer to get rid of rbc's. I am not centrifuging the samples, to avoid loosing the cells. After the lysis I read the samples on FACS Canto using FACS Diva6 software. All further  aanalysis is performed using FlowJo. 

currently, I am concerned with two issues:

1. Debri from rbc lysis. Since I am not centrifuging the samples, I am getting a lot of debri from rbc lysis which is far more into the right shoulder of FSC than I expected it, so it kind of complies over the leukocyte population.  

I am actually concerned if in fact all the rbcs are lysing? I prepared fresh lysis buffer per no-wash rbc lysis, but I am seeing the same results

I have to collect a lot of events to get a decent leukocyte population. To avoid the debri, I started gating on the main leukocyte population and recording only those events during acquisition. I am concerned I may be loosing some cells that fall outside of the gate for the majority of leukocyte population. I was wondering if somebody can suggest a strategy to address that.

2. The size of the beads

the beads I am currently using are 7um which should be the relative size of the leukocyte population. I expected them to appear close on the plot. But in fact, i have to reduce SSC largely to fit the beads on the plot and that really compresses my leukocyte population, which I am not sure if I should worry about yet. I vortex the beads a lot to avoid clumping but the problem seems to persist. 

Any suggestions would be helpful

i will include pictures as soon as

thank you 

I'm going to use mice that have Tet-Off system in transgenic mouse, in which the gene of interest is being induced upon the removal of Doxycycline from the diet. My protocol suggests the following feeding scheme (with Doxycycline in food):

40mg/kg Dox (const diet min 5days)  --> NO Dox (4-5 days) to allow expression of the gene of interest --> 100mg/kg Dox (1day) --> 40mg/kg (const diet)

1) Most of the suppliers offer 200mg/kg Doxycycline food and 40mg/kg has to be ordered as a custom diet. Does anyone have an experience with doxycycline and can comment on wether my feeding scheme is optimal? What food supplier can you reccomend?

2) What should be the optimal time window w/o Dox to allow the complete removal of Dox from the system for the proper expression of the gene of interest? 

3) What is the best way to introduce Dox to stop the expression of the gene of interest after the NO-Dox diet (IP vs 1mg/kg in food)? If IP, what amount of Dox should be injected?